Inhibition of microcompetition with a foreign polynucleotide as treatment of chronic disease

ABSTRACT

Microcompetition between a foreign polynucleotide and a cellular polynucleotide is a risk factor associated with chronic disease. The invention uses this novel discovery to present methods for the treatment of chronic disease. The methods are based on modifying the microcompetition between a polynucleotide natural to a subject suffering from a chronic disease and a polynucleotide foreign to the subject. Specifically, the treatment may modify the cellular copy number of the foreign polynucleotide, change the rate of complex formation between the microcompeted cellular transcription factor and either the foreign polynucleotide or the cellular polynucleotide, vary the expression of a gene susceptible to such microcompetition, or manipulate the activity of a gene product of a gene susceptible to microcompetition with a foreign polynucleotide. The invention also presents methods for the treatment of chronic disease resulting from other foreign polynucleotide-type disruptions.

BACKGROUND OF THE INVENTION

[0001] The cause of many cases of the major chronic diseases is unknown.Therefore, treatment is focused on clinical symptoms associated with thedisease rather than the cause. As a result, in many cases, the treatmentshows limited efficacy and serious negative side effects.

[0002] Recently, the National Cancer Institute (NIH Guide 2000¹)announced a program aimed to “reorganize the “front-end,” or gateway, todrug discovery in cancer. The new approach promotes a three stagediscovery process; first, discovery of the molecular mechanismsunderlying neoplastic transformations, cancer growth and metastasis;second, selection of a novel molecular target within the discoveredbiochemical pathway associated with the disease state; finally, designof a new drug that modifies the selected target. The program encouragesmoving away from screening based on a clinical effects, such as tumorcell shrinkage, either in vivo or in vitro, to screening, or drugdesign, based on molecular effects. According to the NCI, screening by adesired clinical effect identified drugs that traditionally demonstratedclear limitations in patients, while screening by a desired moleculareffect should produce more efficacious and specific drugs.

[0003] The best drugs reverse the molecular events that cause a disease.Following the discovery of microcompetition between foreignpolynucleoitdes and cellular genes as the cause of many chronic diseasecases, the present invention presents methods for treating chronicdiseases, methods for evaluating the effectiveness of a compound for usein modulating the progression of chronic diseases, and methods fordetermining whether a subject has a chronic disease, or has an increasedrisk of developing clinical symptoms associated with such disease.

BRIEF SUMMARY OF THE INVENTION

[0004] In one aspect, the invention presents methods for treatingchronic diseases. In a preferred embodiment, the methods featureadministration to a subject a therapeutically effective amount of apharmaceutical or nutraceutical composition that attenuatesmicrocompetition between a foreign polynucleotide and a cellularpolynucleotide, attenuates an effect of such microcompetition, orattenuates an effect of another foreign polynucleotide-type disruption.A pharmaceutical or nutraceutical composition may include, but notlimited to, small molecule (organic or inorganic), polynucleotide,polypeptide or antibody.

[0005] For example, to ameliorate a disease symptom resulting frommicrocompetition between a foreign polynucleotide and a cellularpolynucleotide, a pharmaceutical composition can be administered to thesubject that reduces the cellular copy number of the foreignpolynucleotide, reduces complex formation between the foreignpolynucleotide and a cellular transcription factor, increases complexformation between the microcompeted cellular transcription factor andthe cellular polynucleotide, or reverses an effect of microcompetitionon the expression or activity of a polypeptide with expression regulatedby the cellular polynucleotide. For example, in the case of a p300/cbpvirus and the cellular Rb gene, a pharmaceutical composition can beadministered to the subject that reduces the copy number of the p300/cbpvirus by, for instance, reducing viral replication, reduces binding of ap300/cbp transcription factor, such as GABP, to the p300/cbp virus,increases expression of the p300/cbp transcription factor, increasesbinding of the p300/cbp transcription factor to the Rb promoter by, forinstance, stimulating phosphorylation of the p300/cbp transcriptionfactor, or increases expression of Rb, through, for instance,transfection of an exogenous Rb gene, reduced degradation of the Rbprotein, or administration of exogenous Rb protein (see more examplesbelow).

[0006] In the case of another foreign polynucleotide-type disruption,for example, the composition may reverse the effects of such disruption.For instance, microcompetition with a p300/cbp virus reduces expressionof Rb. A mutation can also reduce the expression of Rb. Therefore, suchmutation is a foreign polynucleotide-type disruption. Microcompetitionwith a p300/cbp virus can result in cancer, and, therefore, a mutationin the Rb promoter that reduces Rb expression can also result in cancer.To ameliorate the symptoms of cancer resulting from such mutation in theRb gene, a pharmaceutical composition can be administered to the subjectthat stimulates complex formation between a p300/cbp transcriptionfactor and Rb.

[0007] In second aspect, the invention provides assays for screeningtest compounds to find compounds which modulate microcompetition betweena foreign polynucleotide and a cellular polynucleotide, an effect ofsuch microcompetition, or an effect of another foreignpolynucleotide-type disruption.

[0008] A further aspect of the invention provides methods fordetermining the risk of developing the molecular, cellular and clinicalsymptoms associated with a chronic disease. The method may includedetecting in a biological sample obtained from a subject at least one ofthe following: (i) a foreign polynucleotide, specifically, a p300/cbpvirus (ii) modified expression or bioactivity of a gene suceptible tomicrocompetition with a foreign polynuleotide, specifially, a p300/cbpregulated gene (iii) presence of a genetic lesion in a gene suceptibleto microcompetition with a foreign polynculetide, specifically, a geneencoding a p300/cbp factor, a p300/cbp regulated gene, p300/cbp factorkinase or p300/cbp phosphatase, or p300/cbp agent (iv) presence of agenetic lesion in a DNA binding box of a p300/cbp transcription factor.

BRIEF DESCRIPTION OF THE FIGURES

[0009]FIG. 1 shows the observed relative CAT activity as a function ofthe relative concentration of the competitor plasmid pX1.0 to the testplasmid pSV2CAT.

[0010]FIG. 2 shows the observed relative CAT activity as a function ofthe relative concentration of the competitor plasmid pSV2neo to the testplasmid pSV2CAT, and the relative concentration of the competitorplasmid pA10neo to the test plasmid pSV2CAT, in either Ltk- or MLfibroblast cells.

[0011]FIG. 3 shows the observed relative CAT activity, expressed as theratio between CAT activity in the presence of pSV2neo and CAT activityin the absence of pSV2neo, as a function of the molar ratio of pSV2Neoto hMT-IIA-CAT.

[0012]FIG. 4 shows accumulation of triglyceride assayed by oil redstaining in untreated F442A cells or in cells transfected with either avector expressing the SV40 large T antigen or the “empty vector”pZIPNeo.

[0013]FIG. 5 shows the observed relative CAT activity as a function ofthe molar ratio between the competitor plasmid CMV-βgal and the testplasmid PDGF-B-CAT, or between the competitor plasmid SV40-βgal and thetest plasmid PDGF-B-CAT.

[0014]FIG. 6 shows the observed HSL mRNA in undifferentiated confluentcontrols and in differentiated 3T3-L1 cells transfected with the pZipNeovector.

[0015]FIG. 7 shows the observed number of viable cells followingtransfection with either the pBARB vector or the “empty vector” pSV-neo.

[0016]FIG. 8 shows the observed luc activity following transfection with20 ng pIRES-AR, pcDNA-AR or pSG5-AR plasmids which express AR, 500 ngMMTV-luc which highly expresses luc following AR stimulation of the MMTVpromoter, and increasing amounts of the empty expression vector, whereluc activity in the presence of 650 ng pGEM-7Zf(+) was arbitrarily setto 1.

[0017]FIG. 9 shows the observed luc activity following transfection with20 ng pSG5-AR, 20 ng pS40-β-galactosidase (βGAL) and increasing amountsof the empty vector pSG5, where luc and βGAL activities in the presenceof 650 pGEM-7Zf(+) were arbitrarily set to 1.

[0018]FIG. 10 shows the observed number of cells over time followingtransfection with either the pcDNA3 vector carrying an antisense to themacrophage inflammatory protein 1-α (MIP-1α) or with the “empty” pcDNA3vector.

[0019]FIG. 11 shows the observed volume of tumors injected with thevector encoding the icon, volume of uninjected tumors in the icontreated mice, volume of tumors injected with the empty vectorpcDNA3.1(+), and volume of uninjected tumors in the empty vectorinjected mice, over time, following injection of SCID mice s.c. in bothrear flanks with the human prostatic cancer line c4-2.

[0020]FIG. 12 shows the observed volume of tumors injected with thevector encoding the icon, volume of uninjected tumors in the icontreated mice, volume of tumors injected with the empty vectorpcDNA3.1(+), and volume of uninjected tumors in the empty vectorinjected mice, over time, following injection of SCID mice s.c. in bothrear flanks with the human melanoma line TF2.

DETAILED DESCRIPTION OF THE INVENTION A. Introduction of Invention 1.Detailed Description of New Elements

[0021] The following sections present descriptions of elements used inthe present invention. Following each definition, one or more exemplaryassays are provided to illustrate to one skilled in the art how to usethe element. Each assay may include, as its own elements, standardmethods in molecular biology, microbiology, cell biology, cell culture,transgenic biology, recombinant DNA, immunology, pharmacology, andtoxicology, well known in the art. Details of the standard methods areavailable further below.

a) Microcompetition Related Elements

[0022] (1) Microcompetition

[0023] Definition

[0024] Assume the DNA sequences DNA₁ and DNA₂ bind the transcriptioncomplexes C₁ and C₂, respectively. If C₁ and C₂ include the sametranscription factor, DNA₁ and DNA₂ are called “microcompetitors.” Aspecial case of microcompetition is two DNA sequences which bind thesame transcription complex.

[0025] Notes:

[0026] 1. Transcription factors include transcription coactivators.

[0027] 2. Sharing the same environment, such as cell, or chemical mix,is not required to be regarded microcompetitors. For instance, two geneswhich were shown once to bind the same transcription factor are regardedmicrocompetitors independent of their actual physical environment. Toemphasize such independence, the terminology “susceptible tomicrocompetition” may be used.

[0028] Exemplary Assays

[0029] 1. If DNA₁ and DNA₂ are endogenous in the cell of interest, assaythe transcription factors bound to the DNA sequences (see in “Detaileddescription of standard protocols” below, the section entitled“Identifying a polypeptide bound to DNA or protein complex”) and comparethe two sets of polypeptides. If the two sets include a commontranscription factor, DNA₁ and DNA₂ are microcompetitors.

[0030] 2. In assay 1, if DNA₁ and/or DNA₂ are not endogenous, introduceDNA₁ and/or DNA₂ to the cell by, for instance, transfecting the cellwith plasmids carrying DNA₁ and/or DNA₂, infecting the cell with a virusthat includes DNA₁ and/or DNA₂, and mutating endogenous DNA to produce asequence identical to DNA₁ and/or DNA₂.

[0031] Notes:

[0032] 1. Introduction of exogenous DNA₁ and/or DNA₂ is a special caseof modifying the cellular copy number of a DNA sequence. Suchintroduction increases the copy number from zero to a positive number.Generally, copy number may be modified by means such as the onesmentioned above, for instance, transfecting the cell with plasmidscarrying a DNA sequence of interest, infecting the cell with a virusthat includes the DNA sequence of interest, and mutating endogenous DNAto produce a sequence identical to the DNA sequence of interest.

[0033] 2. Assume DNA₁ and DNA₂ microcompete for the transcription factorF. Assaying the copy number of at least one of the two sequences, thatis, DNA₁ and/or DNA₂, is regarded as assaying microcompetition for F,and observing a change in the copy number of at least one of the twosequences is regarded as identification of modified microcompetition forF.

[0034] 3. Assume the transcription factor F binds the DNA box DNA_(F).Consider a specific DNA sequence, DNA₁ which includes a DNA_(F) box,then:

[F•DNA₁]f([DNA_(F)], [F], F-affinity, F-avidity)

[0035] The concentration of F bound to DNA₁ is a function of the DNA_(F)copy number, the concentration of F in the cell, F affinity and avidityto its box. Using f, a change in microcompetition can be defined as achange in [DNA_(F)], and a change in [F•DNA₁] as an effect of suchchange.

[0036] 4. Note that under certain conditions (fixed [F], fixedF-affinity, fixed F-avidity, and limiting transcription factor (seebelow)), there is a “one to one” relation between [F•DNA₁] and[DNA_(F)]. Under such conditions, assaying [F•DNA₁] is regarded assayingmicrocompetition.

[0037] Examples

[0038] See studies in the section below entitled “Microcompetition witha limiting transcription complex.”

[0039] (2) Microavailable

[0040] Definition

[0041] Let L₁ and L₂ be two molecules. Assume L₁ can take s=(1 . . . .n) shapes. Let L_(1,s) denote L₁ in shape s, and let [L_(1,s)] denoteconcentration of L_(1,s). If L_(1,s) can bind L₂, an increase (ordecrease) in [L_(1,s)] in the environment of L₂ is called “increase (ordecrease) in microavailability of L_(1,s) to L₂.” Microavailability ofL_(1,s) is denoted _(ma)L_(1,s). A shape that does not bind L₂ is called“microunavailable to L₂.”

[0042] Let s=(1 . . . m) denote the set of all L_(1,s) that can bind L₂.Any increase (or decrease) in the sum of [L_(1,s)] over all s=(1 . . .m) is called “increase (or decrease) in microavailability of L₁ to L₂.”Microavailability of L₁ to L₂ is denoted _(ma)L₁.

[0043] Notes:

[0044] 1. A molecule in a complex is regarded in a different shaperelative to the same molecule uncomplexed, or free.

[0045] 2. Consider an example of an antibody against L_(1,j), a specificshape of L₁. Assume the antibody binds L_(1,j) in the region contactingL₂. Assume the antibody binds a single region of L_(1,j), and thatantibody binding prevents formation of the L₁•L₂ complex. By bindingL_(1,j), the antibody changes the shape of L₁ from L_(1,j) to L_(1,k)(from exposed to hidden contact region). Since L_(1,k) does not bind L₂,the decrease in [L_(1,j)] decreases _(ma)L₁, or the microavailability ofL₁ to L₂. If, on the other hand, the antibody converts L_(1,j) toL_(1,p), a shape which also forms the L₁•L₂ complex with the sameprobability, _(ma)L₁ is fixed. The decrease in [L_(1,j)] is equal to theincrease in [L_(1,p)], resulting in a fixed sum of [L_(1,s)] computedover all s which bind L₂.

[0046] Exemplary Assays

[0047] The following assays identify a change in _(ma)L₁ followingtreatment.

[0048] 1. Assay in a biological system (e.g., cell, cell lysate,chemical mixture) the concentrations of all L_(1,s) where s is a shapethat can bind L₂. Apply a treatment to the system which may changeL_(1,s). Following that treatment assay again the concentrations of allL_(1,s) where s is a shape that can bind L₂. Calculate the sum of[L_(1,s)] over all s, before and after treatment. An increase (ordecrease) in this sum indicates an increase (or decrease) in_(ma)L₁.

[0049] Examples

[0050] Antibodies specific for L_(1,s) may be used inimmunoprecipitation, Western blot or immunoaffinity to quantify thelevels of L_(1,s) before and after treatment.

[0051] See also examples below.

[0052] (3) Limiting Transcription Factor

[0053] Definition

[0054] Assume the transcription factor F binds DNA₁. F is called“limiting in respect to DNA₁,” if a decrease in microavailability of Fto DNA₁ decreases the concentration of F bound to DNA₁ (“bound F”).

[0055] Notes:

[0056] 1. The definition characterizes “limiting” by the relationshipbetween the concentration of microavailable F and the concentration of Factually bound to DNA₁. According to the definition, “limiting” means adirect relationship between a decrease in microavailable F and adecrease in bound F, and “not limiting” means no such relationshipbetween the two variables. For instance, according to this definition, adecrease in microavailable F with no corresponding change in bound F,means “not limiting.”

[0057] 2. Let G₁ denote a DNA sequence of a certain gene. Such DNAsequence may include coding and non coding regions of a gene, such asexons, introns, promoters, enhancers, or other segments positioned 5′ or3′ to the coding region. Assume the transcription factor F binds G₁. Anassay can measure changes in G₁ mRNA expression instead of changes inthe concentration of bound F. Assume F transactivates G₁. Since F isnecessary for transcription, a decrease in _(ma)F decreases F•G₁, which,in turn, decreases G₁ transcription. However, an increase inconcentration of F bound to G₁ does not necessarily increasetranscription if binding of F is necessary but not sufficient fortransactivation of G₁.

[0058] Exemplary Assays

[0059] 1. Identify a treatment that reduces _(ma)F by trying differenttreatments, assaying _(ma)F following each treatment, and choosing atreatment which reduces _(ma)F. Assay the concentration of F bound toDNA₁ (see “Basic protocols”) in a biological system (e.g. cell ofinterest). Use the identified treatment to reduce _(ma)F. Followingtreatment assay again the concentration of bound F. A decrease in theconcentration of F bound to DNA₁ indicates that F is limiting in respectto DNA₁.

[0060] 2. Transfect a recombinant expression vector carrying the geneexpressing F. Expression of this exogenous F will increase theintracellular concentration of F. Following transfection:

[0061] (a) Assay the concentration of F bound to DNA₁. An increase inconcentration of bound F indicates that F is limiting in respect toDNA₁.

[0062] (b) If DNA₁ is the gene G₁, assay G₁ transcription. An increasein G₁ transcription indicates that F is limiting in respect to G₁ (suchan increase in transcription is expected if binding of F to G₁ issufficient for transactivation).

[0063] 3. Contact a cell with antibodies which reduce _(ma)F. Followingtreatment:

[0064] (a) Assay the concentration of F bound to DNA₁. A decrease inconcentration of bound F with any antibody concentration indicates thatF is limiting in respect to DNA₁.

[0065] (b) If DNA₁ is the gene G₁, assay G₁ transcription. A decrease inG₁ transcription with any antibody concentration indicates that F islimiting in respect to G₁.

[0066] See Kamei 1996² which used anti-CBP immunoglubulin G (IgG).(Instead of antibodies, some studies used E1A, which, by binding top300/cbp, also converts the shape from microavailable tomicrounavailable).

[0067] 4. Modify the copy number of DNA₂, another DNA sequence, or G₂,another gene, which also bind F (by, for instance, transfecting the cellwith DNA₂ or G₂, see above).

[0068] (a) Assay the concentration of F bound to DNA₁. A decrease inconcentration of F bound to DNA₁ indicates that F is limiting in respectto DNA₁.

[0069] (b) If DNA₁ is the gene G₁, assay G₁ transcription. A decrease inG₁ transcription indicates that F is limiting in respect to G₁.

[0070] If DNA, is the gene G₁, competition with DNA₂ or G₂, which alsobind F, reduces the concentration of F bound to G₁ and, therefore, theresulting transactivation of G₁ in any concentration of DNA₂ or G₂. Inrespect to G₁, binding of F to DNA₂ or G₂ reduces microavailability of Fto G₁, since F bound to DNA₂ or G₂ is microunavailable for binding withG₁.

[0071] This assay is exemplified in a study reported by Kamei 1996(ibid). The study used TPA to stimulate transcription from a promotercontaining an AP-1 site. AP-1 interacts with CBP. CBP also interactswith a liganded retinoic acid receptor (RAR) and liganded glucocorticoidreceptor (GR) (Kamei 1996, ibid, FIG. 1). Both RAR and GR exhibitedligand-dependent repression of TPA stimulated transcription. Inductionby TPA was about 80% repressed by treatment with retinoic acid ordexamethasone. In this study, G is the gene controlled by the AP-1promoter. In respect to this gene, the CBP•liganded-RAR complex is themicrounavailable form. An increase in [CBP•liganded-RAR] decreases theconcentration of microavailable CBP.

[0072] In another exemplary study by Hottiger 1998³, the two genes areHIV-CAT, which binds NF-κB, and GAL4-CAT, which binds the fusion proteinGAL4-Stat2(TA). NF-κB binds p300/cbp. The GAL4-Stat2(TA) fusion proteinincludes the Stat2 transactivation domain which also binds p300/cbp. Thestudy showed a close dependent inhibition of gene activation by thetransactivation domain of Stat2 following transfection of a RelAexpression vector (Hottiger 1998, ibid, FIG. 6A).

[0073] 5. Transfect F and modify the copy number of DNA₂, another DNAsequence, or G₂, a another gene, which also bind F (by, for instance,transfecting the cell with DNA₂ or G₂, see also above). Followingtransfection:

[0074] (a) Assay concentration of F bound to DNA₁. Attenuated decreasein concentration of F bound to DNA₁ indicates that F is limiting inrespect to DNA₁.

[0075] (b) If DNA₁ is the gene G₁, assay G₁ transcription. Attenuateddecrease in G₁ transactivation caused by DNA₂ or G₂, indicates that F islimiting in respect to G₁ (see Hottiger 1998, ibid, FIG. 6D).

[0076] 6. Call the box which binds F the “F-box.” Transfect a cell withDNA₂, another DNA sequence, or G₂, another gene, carrying a wild typeF-box. Transfect another cell with with DNA₂ or G₂, after mutating theF-box in the transfected DNA₂ or G₂.

[0077] (a) Assay the concentration of F bound to DNA₁. Attenuateddecrease in the concentration of F bound to DNA₁ with the wild type butnot the mutated F-box indicates that F is limiting in respect to DNA₁.

[0078] (b) If DNA1 is the gene G₁, assay G₁ transcription. Attenuateddecrease in G₁transactivation with the wild type but not the mutatedF-box indicates that F is limiting in respect to G₁.

[0079] If DNA1 is the gene G₁, a mutation in the F-box results indiminished binding of F to DNA₂ or G₂, and an attenuated inhibitoryeffect on G₁ transactivation. In Kamei 1996 (ibid), mutations in the RARAF2 domain that inhibit binding of CBP, and other coactivator proteins,abolished AP-1 repression by nuclear receptors.

[0080] 7. Let t₁ and t₂ be two transcription factors which bind F. LetG₁ and G₂ be two genes transactivated by the t₁•F and t₂•F complexes,respectively.

[0081] (a) Transfect a cell of interest with t₁ and assay G₂transcription. If the increase in [t₁] reduces transcription of G₂, F islimiting in respect to G. Call t₂•F the microavailable shape of F inrespect to G₂. The increase in [t₁] increases [t₁•F], which, in turn,reduces [t₂•F]. The decrease in the shape of F microavailable to G₂reduces transactivation of G₂. In Hottiger 1998 (ibid), t₁ is RelA, t₂is GAL4-Stat2(TA) and G₂ is GAL4-CAT. See results of the increase in t₁on G₂ transactivation shown in Hottiger (1998, ibid) FIG. 6A.

[0082] (b) Transfect F and assay the concatenation of F bound to G, ortransactivation of G. If the increase in F decreases the inhibitoryeffect of t₁, F is limiting in respect to G (see Hottiger 1998 (ibid),FIG. 6C showing the effect of p300/cbp transfection).

[0083] (c) Assay the concentration of t₁, t₂ and F. If t₁ and t₂ have ahigh molar excess compared to F, F is limiting in respect to G (seeHottiger 1998, (ibid)).

[0084] (4) Microcompetition for a Limiting Factor

[0085] Definition

[0086] Assume DNA₁ and DNA₂ microcompete for the transcription factor F.If F is limiting in respect to DNA₁ and DNA₂, DNA₁ and DNA₂ are called“microcompetitiors for a limiting factor.”

[0087] Exemplary Assays

[0088] 1. The assays 4-7 in the section entitled “Limiting transcriptionfactor” above, can be used to identify microcompetition for a limitingfactor.

[0089] 2. Modify the copy number of DNA₁ and DNA₂ (by, for instance,co-transfecting recombinant vector carrying DNA₁ and DNA₂, see alsoabove).

[0090] (a) Assay DNA₁ protection against enzymatic digestion (“DNasefootprint assay”). A change in protection indicates microcompetition fora limiting factor.

[0091] (b) Assay DNA, electrophoretic gel mobility (“electrophoreticmobility shift assay”). A change in mobility indicates microcompetitionfor a limiting factor.

[0092] 3. If DNA, is a segment of a promoter or enhancer, or canfunction as a promoter or enhancer, independently, or in combination ofother DNA sequences, fuse DNA, to a reporter gene such as CAT or LUC.Co-transfect the fused DNA₁ and DNA₂. Assay for expression of thereporter gene. Specifically, assay transactivation of reporter genefollowing an increase in DNA₂ copy number. A change in transactivationof the reporter gene indicates microcompetition for a limiting factor.

[0093] 4. A special case is when DNA₁ is the entire cellular genomeresponsible for normal cell morphology and function. Transfect DNA₂, andassay cell morphology and/or function (such as, binding of extracellularprotein, cell replication, cellular oxidative stress, genetranscription, etc). A change in cell morphology and/or functionindicates microcompetition for a limiting factor.

[0094] Notes:

[0095] 1. Preferably, following co-transfection of DNA₁ and DNA₂, verifythat the polynucleotides do not produce mRNA. If the sequencestranscribe mRNA, block translation of proteins with, for instance, anantisense oligonucleotide specific for the exogenous mRNA.Alternatively, verify that the proteins are not involved in binding of Fto either sequence. Also, verify that co-transfection does not mutatethe F-boxes in DNA₁ and DNA₂, and that the sequences do not change themethylation patterns of their F-boxes. Finally, check that DNA₁ and DNA₂do not contact each other in the F-box region.

[0096] Examples

[0097] See studies in the section below entitled “Microcompetition witha limiting transcription complex.”

[0098] (5) Foreign to

[0099] Definition 1

[0100] Consider an organism R with standard genome O. Consider O_(s) asegment of O. If a polynucleotide Pn is different from O_(s) for allO_(s) in O, Pn is called “foreign to R.”

[0101] Notes:

[0102] 1. As an example for different organisms consider the list ofstandard organisms in the PatentIn 3.1 software. The list includesorganisms such as, homo sapiens (human), mus musculus (mouse), ovisaries (sheep), and gallus gallus (chicken).

[0103] 2. A standard genome is the genome shared by most representativesof the same organism.

[0104] 3. A polynucleotide and DNA sequence (see above) areinterchangeable concepts.

[0105] 4. In multicellular organism, such as humans, the standard genomeof the organism is not necessarily found in every cell. The genomesfound in sampled cells can vary as a result of somatic mutations, viralintegration, etc (see definition below of foreign polynucleotide in aspecific cell).

[0106] 5. Assume Pn expresses the polypeptide Pp. If Pn is foreign to R,then Pp is foreign to R. 6. When the reference organism is evident,instead of the phrase “a polynucleotide foreign to organism R,” the“foreign polynucleotide” phrase might be used.

[0107] Exemplary Assays

[0108] 1. Compare the sequence of Pn with the sequence, or sequences ofthe published, or self sequenced standard genome of R. If the sequenceis not a segment of the standard genome, Pn is foreign to R.

[0109] 2. Isolate DNA from O (for instance, from a specific cell, or avirus). Try to hybridize Pn to the isolated DNA. If Pn does nothybridize, it is foreign.

[0110] Notes:

[0111] 1. Pn can still be foreign if it hybridizes with DNA from aspecific O specimen. Consider, for example, the case of integrated viralgenomes. Viral sequences integrated into cellular genomes are foreign.To increase the probability of correct identification, repeat the assaywith N>1 specimens of O (for instance, by collecting N cells fromdifferent representatives of R). Define the genome of R as all DNAsequences found in all O specimens. Following this definition,integrated sequences which are only segments of certain O specimens areidentified as foreign. Note that the test is dependent on the Npopulation. For instance, a colony which propagates from a single cellmight include a foreign polynucleotide in all daughter cells. Therefore,the N specimens should include genomes (or cells) from differentlineages.

[0112] 2. A polynucleotide can also be identified as potentially foreignif it is found episomally in the nucleus. If the DNA is found in thecytoplasm, it is most likely foreign. Also, a large enoughpolynucleotide can be identified as foreign if many copies of thepolynucleotide can be observed in the nucleus. Finally, if Pn isidentical to sequences in genomes of other organisms, such as viruses orbacteria, known to invade R cells, and specifically nuclei of R cells,Pn is likely foreign to R.

[0113] Definition 2

[0114] Consider an organism R. If a polynucleotide Pn is immunologicallyforeign to R, Pn is called “foreign to R.”

[0115] Notes:

[0116] 1. In Definition 1, the comparison between O, the genome of the Rorganism, and Pn is performed logically by the observer. In definition2, the comparison is performed biologically by the immune system of theorganism R.

[0117] 2. Definition 2 can be generalized to any compound or substance.A compound X is called foreign to organism R, if X is immunologicallyforeign to R.

[0118] Exemplary Assays

[0119] 1. If the test polynucleotide includes a coding region,incorporate the test polynucleotide in an expressing plasmid andtransfer the plasmid into organism R, through, for instance, injection(see DNA-based immunization protocols). An immune response against theexpressed polypeptide indicates that the polynucleotide is foreign.

[0120] 2. Inject the test polynucleotide in R. An immune responseagainst the injected polynucleotide indicates that the testpolynucleotide is foreign.

[0121] Examples

[0122] Many viruses, nuclear, such as Epstein-Barr, and cytoplasmic,such as Vaccinia, express proteins which are antigenic and immunogenicin their respective host cells.

[0123] Definition 3

[0124] Consider an organism R with standard genome O. Consider O_(s), asegment of O. If a polynucleotide Pn is chemically or physicallydifferent than O_(s) for all O, in O, Pn is called “foreign to R.”

[0125] Notes:

[0126] 1. In Definition 3, the observer compares O, the genome of the Rorganism, with Pn using the molecules chemical or physicalcharacteristics.

[0127] Exemplary Assays

[0128] In general, many assays in the “Detection of a genetic lesion”section below compare a test polynucleotide and a wild-typepolynucleotide. In these assay, let O_(s) be the wild-typepolynucleotide and use the assays to identify a foreign polynucleotide.Consider the following examples.

[0129] 1. Compare the electrophoretic gel mobility of O_(s) and the testpolynucleotide. If mobility is different, the polynucleotides aredifferent.

[0130] 2. Compare the patterns of restriction enzyme cleavage of O_(s)and the test polynucleotide. If the patterns are different, thepolynucleotides are different.

[0131] 3. Compare the patterns of methylation of O_(s) and the testpolynucleotide (by, for instance, electrophoretic gel mobility). If thepatterns are different, the polynucleotides are different.

[0132] Definition 4

[0133] Consider an organism R with standard genome O. Let [Pn] denotethe copy number of Pn in O. Consider a cell Cell_(i). Let [Pn]_(i)denote the copy number of Pn in Cell_(i). If [Pn]_(i)>[Pn], Pn is called“foreign to Cell_(i).”

[0134] Note

[0135] 1. [Pn]_(i) is the copy number of all Pn in Cell_(i), from allsources. For instance, [Pn] includes all Pn segments in O, all Pnsegments of viral DNA in the cell (if available), all Pn segments ofplasmid DNA in the cell (if available), etc.

[0136] 1. If [Pn]=0, the definition is identical to definition 1 offoreign polynucleotide.

[0137] Exemplary Assays

[0138] 1. Sequence the genome of Cell_(i). Count the number of time Pnappears in the genome. Compare the result to the number of times Pnappears in the published standard genome. If the number is greater, Pnis foreign to Cell_(i).

[0139] 2. Sequence the genome of Cell_(i) and a group of other cellsCell_(j) . . . , Cell_(j+m). If [Pn]_(i)>[Pn]_(j)= . . . =[Pn]_(j+m), Pnis foreign to Cell_(i).

[0140] (6) Natural to

[0141] Definition

[0142] Consider an organism R with standard genome O. If apolynucleotide Pn is a fragment of O, Pn is called “natural to R.”

[0143] Notes:

[0144] 1. “Natural to” and “foreign to” are mutually exclusive. Apolynucleotide cannot be both foreign and natural to R. If apolynucleotide is natural, it is not foreign to R, and if apolynucleotide is foreign, it is not natural to R.

[0145] 2. If Pn is a gene natural to R, then, the its gene product isalso natural to R.

[0146] 3. The products of a reaction carried out in a cell between geneproducts natural to the cell, under normal conditions, are natural tothe cell. For instance, cellular splicing by factors natural to the cellproduce splice products natural to the cell.

[0147] Exemplary Assays

[0148] 1. Compare the sequence of Pn with the sequence, or sequences ofthe published, or self sequenced standard genome of R. If the sequenceis a segment of the standard genome, Pn is natural to R.

[0149] 2. Isolate DNA from O (for instance, from a specific cell, or avirus). Try to hybridize Pn to the isolated DNA. If Pn hybridizes, it isnatural.

[0150] Notes:

[0151] 1. Hybridization with DNA from a specific O specimen of R is notconclusive evidence that Pn is natural to R. Consider, for example, thecase of integrated viral genomes. Viral sequences integrated intocellular genomes are foreign. To increase the probability of correctidentification, repeat the assay with N>1 specimens of O (for instance,by collecting N cells from different representatives of R). Define thegenome of R as all DNA sequences found in all O specimens. Followingthis definition, integrated sequences which are only segments of certainO specimens are identified as foreign. Note that the test is dependenton the N population. For instance, a colony which propagates from asingle cell might include a foreign polynucleotide in all daughtercells. Therefore, the N specimens should include genomes (or cells) fromdifferent lineages.

[0152] (7) Empty Polynucleotide

[0153] Definition

[0154] Consider the Pn polynucleotide. Consider an organism R withgenome O_(R). Let Pp(Pn), and Pp(O_(R)) denote a gene product(polypeptide) of a Pn or O_(R) gene, respectively. If Pp(Pn)≠Pp(O_(R))for all Pp(Pn), Pn will be called an “empty polynucleotide” in respectto R.

[0155] Notes:

[0156] 1. A vector is a specific example of a polynucleotide.

[0157] 2. A vector that includes a non coding polynucleotide natural toR is considered empty in respect to the R. (“natural to” is the oppositeof “foreign to.” Note: A natural polynucleotide means, a polynucleotidenatural to at least one organism. An artificial polynucleotide means apolynucleotide foreign to all known organisms. A viral enhancer is anatural polynucleotide. A plasmid with a viral enhancer fused to a humangene is artificial.)

[0158] 3. A vector that includes a coding gene natural to Q, an organismdifferent from R, can still be considered empty in respect to R. Forinstance, a vector that includes the bacterial chloramphenicoltransacetylase (CAT), bacterial neomycin phosphotransferase (neo), orthe firefly luciferase (LUC) as reporter genes, but no human coding geneis considered empty in respect to the humans if it does not express agene natural to humans.

[0159] Exemplary Assays

[0160] 1. Identify all gene products encoded by Pn. Compare to the geneproducts of O_(R). If all gene products are different, Pn is consideredempty in respect to the R.

[0161] Examples pSV2CAT, which expresses the chloramphenicolacethyltransferase (CAT) gene under the control of the SV40promoter/enhancer, pSV2neo, which expresses the neo gene under thecontrol of the SV40 promoter/enhancer, HSV-neo, which expresses theneomycin-resistance gene under control of the murine Harvey sarcomavirus long terminal repeat (LTR), pZIP-Neo, which expresses theneomycin-resistant gene under control of the Moloney murine leukemiavirus long terminal repeat (LTR), are considered empty polynucleotides,or empty vectors, in respect to humans and in respect to the respectivevirus. See more examples below.

[0162] Note: These vectors can be considered as “double” empty, empty inrespect to humans, and empty in respect to the respective virus.

[0163] (8) Latent Foreign Polynucleotide

[0164] Definition

[0165] Consider Pn, a polynucleotide foreign to organism R. Pn will becalled latent in a Cell_(i) of R if over an extended period of time,either:

[0166] 1. Pn produces no Pn transcripts.

[0167] 2. Denote the set of gene products expressed by Pn in Cell_(i)with Cell_(i) _(—) Pp(Pn) and the set of all possible gene products ofPn with All_Pp(Pn), then, Cell_(i) _(—) Pp(Pn) ⊂ All_Pp(Pn), that is,the set of Pn gene products expressed in Cell_(i) is a subset of allpossible Pn gene products.

[0168] 3. Pn shows limited or no replication.

[0169] 4. Pn is undetected by the host immune system.

[0170] 5. Cells shows no lytic symptoms.

[0171] 6. R shows no macroscopic symptoms.

[0172] Notes:

[0173] 1. A virus in a host cell is a foreign polynucleotide. Accordingto the definition, a virus is considered latent if, over an extendedperiod of time, it either shows partial expression of its gene products,no viral mRNA, limited or no replication, is undetected by the hostimmune system, causes no lytic symptoms in the infected cell, or causesno macroscopic symptoms in the host.

[0174] 2. The above list of characterizations is not exhaustive. Themedical literature includes more aspects of latency that can be added tothe definition.

[0175] Exemplary Assays

[0176] 1. Introduce, or identify a foreign polynucleotide in a hostcell. Assay the polynucleotide replication, or transcription, or mRNA,or gene products over an extended period of time. If the polynucleotideshows limited replication, no transcription, or a limited set oftranscripts, the polynucleotide is latent.

[0177] 2. Introduce, or identify a foreign polynucleotide in a hostcell. Assay the cell over an extended period of time, if the cell showsno lytic symptoms, the polynucleotide is latent.

[0178] Examples

[0179] Using PCR, a study (Gonelli 2001⁴) observed persistent presenceof viral human herpes virus 7 (HHV-7) DNA in biopsies from 50 patientswith chronic gastritis. The study also observed no U14, U17/17, U31, U42and U89/90, HHV-7 specific transcripts highly expressed duringreplication. Based on these observations, the study concluded that“gastric tissue represents a site of HHV-7 latent infection andpotential reservoir for viral reactivation.” To test the effect oftreatment on the establishment of latent herpes simplex virus, type 1(HSV-1) in sensory neurons, another study (Smith 2001⁵) assays theexpression of the latency-associated transcript (LAT), the only regionof the viral genome transcribed at high levels during the period ofviral latency. A recent review (Young 2000⁶) discusses the limited setsof Epstein-Barr viral (EBV) gene products expressed during the period ofviral latency.

[0180] (9) Partial Description

[0181] Definition

[0182] Let C_(i) be a characteristic of a system. Let the set Ci, i=(1 .. . m) be the set of characteristics providing a complete description ofthe system. Any subset of Ci, i=(1 . . . m) is called a “partialdescription” of the system.

[0183] Exemplary Assays

[0184] 1. Chose any set of characteristics describing the system andassay these characteristics.

[0185] Examples

[0186] Assaying blood pressure, blood triglycerides, glucose tolerance,body weight, etc.

[0187] (10) Equilibrium

[0188] Definition

[0189] If a system persists in a state St₀ over time, St₀ is calledequilibrium.

[0190] Note:

[0191] The system related definitions can be modified to accommodatepartial descriptions. For example, consider a description of a systemwhich includes only a proper subset of Ci, i=(1 . . . m). If the valuesmeasured for the subset of characteristics in St₀ persist over time, theprobability that St₀ is an equilibrium is greater than zero. However,since the values are measured only on a subset of Ci, i=(1 . . . m), theprobability is less than 1. Overall, an increase in the size of thesubset of characteristics increases the probability.

[0192] Exemplary Assays

[0193] 1. Assay the values of the complete (sub) set of the systemcharacteristics. Repeat the assays over time. If the values persist, thesystem is (probably) in equilibrium.

[0194] Examples

[0195] Regular physicals include standard tests, such as blood count,cholesterol levels, HDL cholesterol, triglycerides, kidney functiontests, thyroid function tests, liver function tests, minerals, bloodsugar, uric acid, electrolytes, resting electrocardiogram, an exercisetreadmill test, vision testing, and audiometry. When the values in thesetests remain within a narrow range over time, the medical condition ofthe subject can be labeled as a probable equilibrium. Other testsperformed to identify deviations from equilibrium are mammograms andprostate cancer screenings.

[0196] (11) Stable Equilibrium

[0197] Definition

[0198] Consider an equilibrium E₀. If, after small disturbances, thesystem always returns to E₀, the equilibrium is called “stable.” If thesystem moves away from E₀ after small disturbances, the equilibrium iscalled “unstable.”

[0199] Exemplary Assays

[0200] 1. Take a biological system (e.g., cell, whole organism, etc).Assay a set of characteristics. Verify that the system is inequilibrium, that is, the values of these characteristics persist overtime. Apply treatment to the system and assay the set of characteristicsagain. Repeat assaying over time. If the treatment changed the values ofthe characteristics, and within a reasonable time the values returned tothe original levels, the equilibrium is stable.

[0201] (12) Chronic Disease

[0202] Definition

[0203] Let a healthy biological system be identified with a certainstable equilibrium. A stable equilibrium different from the healthysystem equilibrium is called “chronic disease.”

[0204] Notes:

[0205] 1. In chronic disease, in contrast to acute disease, the systemdoes not return to the healthy equilibrium on its own.

[0206] Exemplary Assays

[0207] 1. Take a biological system (e.g., cell, whole organism, etc).Assay a set of characteristics. Compare the results with the values ofthe same characteristics in healthy controls. If some values deviatefrom the values of healthy controls, and the values continue to deviateover time, the equilibrium of the system can be characterizes as chronicdisease.

[0208] Examples

[0209] High blood pressure, high body weight, hyperglycemia, etc.

[0210] (13) Disruption

[0211] Definition

[0212] Let a healthy biological system be identified with a certainstable equilibrium. Any exogenous event which produces a new stableequilibrium is called “disruption.”

[0213] Notes:

[0214] 1. Using the above definitions it can be said that a disruptionis an exogenous event that produces a chronic disease.

[0215] 2. A disruption is a disturbance with a persisting effect.

[0216] Exemplary Assays

[0217] 1. Take a biological system (e.g., cell, whole organism, etc).Assay a set of characteristics. Compare the results with the values ofthe same characteristics in healthy controls. Verify that the system isin healthy equilibrium. Apply a chosen treatment to the system.Following treatment, assay the same characteristics again. If somevalues deviate from the values of healthy controls, continue to assaythese characteristics over time. If the values continue to deviate overtime, the treatment produced a chronic disease, and, therefore, can beconsidered a disruption.

[0218] Examples

[0219] Genetic knockout, carcinogens, infection with persistent viruses(e.g., HIV, EBV), etc.

[0220] (14) Foreign Polynucleotide-Type Disruption (Cause of Disruption)

[0221] Definition

[0222] Let Pp be a polypeptide. Assume microcompetition with a foreignpolynucleotide Pn directly, or indirectly reduces (or increases) Ppbioactivity. A disruption that directly, or indirectly reduces (orincreases) Pp bioactivity is called “foreign polynucleotide-typedisruption.”

[0223] Notes:

[0224] 1. The first “indirectly” in the definition means that Pp can bedownstream from the gene microcompeting with Pn. The second “indirectly”means that Pp can be downstream from the gene, or polypeptide, directlyaffected by the exogenous event. According to the definition, if bothmicrocompetition with a foreign polynucleotide and an exogenous eventincrease, or both decrease bioactivity of Pp, the exogenous event can beconsidered as a foreign polynucleotide-type disruption.

[0225] 2. Microcompetition with a foreign polynucleotide is a specialcase of foreign polynucleotide-type disruption.

[0226] 3. Treatment is a special case of an exogenous event.

[0227] 4. A foreign polynucleotide-type disruption can first affect agene or a polypeptide. For instance, a mutation is an effect on a gene.Excessive protein phosphorylation is an effect on a polypeptide.

[0228] Exemplary Assays

[0229] 1. Take a biological system (e.g., cell, whole organism, etc).Assay a set of characteristics. Compare the results with the values ofthe same characteristics in healthy controls to verify that the systemis in a healthy equilibrium. Modify the copy number of Pn, apolynucleotide of interest (by, for instance, transfection, infection,mutation, etc, see above). Identify a gene with modified expression.Assume the assays show decreased expression of G. Take another specimenof the system in healthy equilibrium and apply a chosen treatment to thehealthy specimen. Following treatment, assay G expression. Continue toassay G expression over time. If G expression is persistently decreased,the exogenous event can be considered a foreign polynucleotide-typedisruption.

[0230] Examples

[0231] A mutation in the leptin receptor, a mutation in the leptin gene,etc (see more examples below).

[0232] (15) Disrupted (Gene, Polypeptide) (Result of Disruption)

[0233] Definition

[0234] Let Pp be a polypeptide. If a foreign polynucleotide-typedisruption modifies (reduces or increases) Pp bioactivity, Pp and thegene encoding Pp are called “disrupted.”

[0235] Notes:

[0236] 1. Pp can be downstream from G, the microcompeted gene.

[0237] Exemplary Assays

[0238] 1. Take a biological system (e.g., cell, whole organism, etc).Modify the copy number of Pn, a polynucleotide of interest, (by, frominstance, transfection, infection, mutation, etc, see above). Assaybioactivity of genes and polypeptides in the treated system and controlsto identify genes and polypeptides with modified bioactivity relative tocontrols. These genes and polypeptides are disrupted.

[0239] Examples

[0240] See studies in the section below entitled “Microcompetition witha limiting transcription complex.” See also all GABP regulated genesbelow.

[0241] (16) Disrupted Pathway

[0242] Definition

[0243] Let the polypeptide Pp_(x) be disrupted. A polypeptide Pp_(i)which functions downstream or upstream of Pp_(x), and the gene encodingPp_(i), are considered a polypeptide and gene, respectively, in a Pp_(x)“disrupted pathway.”

[0244] Exemplary Assays

[0245] 1. Take a biological system (e.g., cell, whole organism, etc).Apply a treatment to the system that modifies Pp_(i) bioactivity. AssayPp_(x) bioactivity. If the bioactivity of Pp_(x) changed, Pp_(i) is in aPp_(x) disrupted pathway.

[0246] 2. Take a biological system (e.g., cell, whole organism, etc).Apply a treatment to the system that modifies Pp_(x) bioactivity. AssayPp_(i) bioactivity. If the bioactivity of Pp_(i) changed, Pp_(i) is in aPp_(x) disrupted pathway.

[0247] Examples

[0248] See examples below.

[0249] (17) Disruptive Pathway

[0250] Definition

[0251] Consider a polypeptide Pp_(k) and a foreign polynucleotide Pn. Ifa change in bioactivity of Pp_(k) increases or decreases Pn copy number,Pp_(k) and the gene encoding Pp_(k) are considered a polypeptide and agene in a Pn “disruptive pathway.”

[0252] Notes:

[0253] Consider, as an example, microcompetition between a cell and aviral polynucleotide, including the entire viral genome. Pp_(k) can beany viral or cellular protein which increase or decreases viralreplication.

[0254] Exemplary Assays

[0255] 1. Take a biological system (e.g., cell, whole organism, etc).Apply a treatment to the system that modifies Pp_(k) bioactivity, forinstance, by increasing expression of a foreign or cellular geneencoding Pp_(k). Assay Pn copy number. If the copy number changed,Pp_(k) and the gene encoding Pp_(k), are in a Pn disruptive pathway.

[0256] Examples

[0257] Consider a GABP virus. The viral proteins which increase viralreplication increase the copy number of viral N-boxes in infected cells.According to the definition, these proteins belong to a disruptivepathway. See specific examples below.

b) p300/cbp Related Elements

[0258] (1) p300/cbp

[0259] Definition

[0260] A member of the p300/cAMP response element (CREB) binding protein(CBP) family of proteins is called p300/cbp.

[0261] Notes:

[0262] 1. For reviews on the p300/cbp family of proteins, see, forinstance, Vo 2001⁷, Blobel 2000⁸, Goodman 2000⁹, Hottiger 2000¹⁰,Giordano 1999¹¹, Eckner 1996¹².

[0263] 2. CREB binding protein (CBP, or CREBBP) is also called RTS,Rubinstein-Taybi syndrome protein, and RSTS.

[0264] 3. See sequences of p300/cbp genes and p300/cbp proteins in theList of Sequences below.

[0265] Exemplary Assays

[0266] 1. p300/cbp may be identified using antibodies in binding assays,oligonucleotide probes in hybridization assays, transcription factorssuch as GABP, NF-κB, E1A in binding assays, etc. (see protocols forbinding and hybridization assays below).

[0267] Examples

[0268] See examples of below.

[0269] (2) p300/cbp Polynucleotide

[0270] Definition

[0271] Assume the polynucleotide Pn binds the transcription complex C.If C contains p300/cbp, Pn is called “p300/cbp polynucleotide.”

[0272] Exemplary Assays

[0273] 1. Take a cell of interest. Modify the copy number of Pn (by, forinstance, transfection, infection, mutation, etc, see also above). Useassays described in the section entitled “Identifying a polypeptidebound to DNA or protein complexes,” or similar assays, to test if theprotein-Pn complexes contain p300/cbp.

[0274] 2. See more assays below.

[0275] Examples

[0276] See below in p300/cbp virus and p300/cbp regulated gene.

[0277] (3) p300/cbp Factor

[0278] Definition

[0279] Assume the transcription factor F binds the complex C. If Ccontains p300/cbp, F is called “p300/cbp factor.”

[0280] Exemplary Assays

[0281] 1. Use assays describe in the section entitled “Identifying apolypeptide bound to DNA or protein complexes,” or similar assays, totest whether the complexes which contain F also contain p300/cbp.

[0282] Examples

[0283] The following table lists some cellular and viral p300/cbpfactors. p300/cbp Gene factor symbol Other names References CellularAML1 RUNX1 acute myeloid leukemia 1 pro- Kitabayashi CBFA2 tein (AML1);core-binding fac- 1998¹³ AML1 tor α2 subunit (CBFα2); onco- gene AML-1;Polyomavirus enhancer binding protein 2αB subunit (PEBP2αB); PEA2αB;SL3-3 enhancer factor 1, αB subunit; SL3/AKV core-binding factor αBsubunit; SEF1; runt- related transcription factor 1; RUNX1; CBFA2 A-MybMYBL1 Myb-related protein A; v-myb Facchinetti AMYB avian myeloblastosisviral onco- 1997¹⁴ gene homolog-like 1 ATF1 ATF1 activatingtranscription factor 1 Goodman TREB36 (ATF1); TREB36 protein; 2000(ibid) cAMP-dependent transcription factor ATF-1 ATF2 ATF2 Activatingtranscription factor 2 Goodman CREB2 (ATF2); cAMP response ele- 2000(ibid), CREBP1 ment binding protein 1 (CRE- Duyndam BP1); HB16;cAMP-dependent 1999¹⁵ transcription factor ATF-2; TREB7; CREB2 ATF4 ATF4activating transcription factor 4 Goodman CREB2 (ATF4); DNA-bindingprotein 2000 (ibid), TAXREB67 TAXREB67; tax-responsive en- Yukawa hancerelement B67 1999¹⁶ (TAXREB67); TXREB; cAMP response element-binding pro-tein 2 (CRBB2); cAMP- dependent transcription factor ATF-4;CCAAT/enhancer bind- ing protein related activating transcription factor(mouse); ApCREB2 (Aplysia) BRCA1 BRCA1 Breast cancer type 1 suscepti-Goodman PSCP bility protein (BRCA1) 2000 (ibid) C/EBPβ CEBPBCCAAT/enhancer binding pro- Goodman TCF5 tein β (C/EBPβ); nuclear factor2000 (ibid), NF-IL6 (NFIL6); transcription Mink 1997¹⁷ factor 5; CRP2;LAP; IL6DBP; CEBPB; TCF5 c-Fos FOS proto-oncogene protein c-fos; GoodmanG0S7 cellular oncogene fos; G0/G1 2000 (ibid), switch regulatory protein7; Sato 1997 v-fos FBJ murine osteosarcoma (ibid) viral oncogenehomolog; FOS; G0S7 C2TA MHC2TA MHC class II transactivator; GoodmanCIITA MHC2TA; CIITA 2000 (ibid), C2TA Sisk 2000 (ibid) AP1 JUNtranscription factor AP-1; proto- Goodman oncogene c-Jun (c-Jun); p39;2000 (ibid), v-jun avian sarcoma virus 17 Hottiger oncogene homolog 2000(ibid) c-Myb MYB Myb proto-oncogene protein; Goodman MYB; v-myb avianmyeloblast- 2000 (ibid), osis viral oncogene homolog Hottiger 2000(ibid) CREB CREB1 cAMP-respone-element-binding Hottiger protein (CREB)2000 (ibid) CRX CRX cone-rod homeobox (CRX); Yanagi CORD2 CRD; cone roddystrophy 2 2000¹⁸ CRD (CORD2) CID CI-D cubitus interruptus dominantGoodman (CID) 2000 (ibid) DBP DBP D-site binding protein (DBP);Lamprecht albumin D box-binding protein; 1999¹⁹ D site of albuminpromoter (albumin D-box) binding protein; TAXREB302 E2F1 E2F1retinoblastoma binding protein 3 Goodman RBBP3 (RBBP-3); PRB-bindingprotein 2000 (ibid), E2F-1; PBR3; retinoblastoma- Marzio associatedprotein 1 (RBAP-1) 2000²⁰ E2F2 E2F2 transcription factor E2F2 Marzio2000 (ibid) E2F3 E2F3 transcription factor E2F3; Marzio 2000 KIAA0075KIAA0075 (ibid) Egr1 EGR1 early-growth response factor-1 SilvermanZNF225 (Egr1); Krox-24 protein; 1998²¹ ZIF268; nerve growth factor-induced protein A; NGFI-A; transcription factor ETR103; zinc fingerprotein 225 (ZNF225); AT225; TIS8; G0S30; ZIF-268 ELK1 ELK1 ets-domainprotein ELK-1 Hottiger 2000 (ibid) ERα ESR1 estrogen receptor α (ERα);Kim 2001²², NR3A1 estrogen receptor 1; estradiol Wang ESR receptor2001²³ , Speir 2000²⁴, Hottiger 2000 (ibid) ERβ ESR2 estrogen receptorβ; ESR2; Kobayashi NR3A2 NR3A2; ESTRB 2000²⁵ ESTRB ER81 Etstranslocation variant 1 Papoutso- (ETV1) poulou 2000²⁶ Ets1 ETS1 C-ets-1protein; v-ets avian Goodman erythroblastosis virus E2 onco- 2000(ibid), gene homolog 1; p54 Jayaraman 1999²⁷ Ets2 ETS2 C-ets-2 protein;human erythro- Jayaraman blastosis virus oncogene homo- 1999 (ibid) log2; v-ets avian erythro- blastosis virus E2 oncogene homolog 2 GABPαGABPA GA binding protein, α subunit Bannert E4TF1A (GABPA); GABP-alphasubunit; 1999²⁸ transcription factor E4TF1-60; nuclear respiratoryfactor-2 subunit alpha (NRF-2A) GABPβ1 GABPB1 GA binding protein beta-1chain Bannert GABPB (GABPB1); GABP-beta-1 sub- 1999 (ibid) E4TF1B unit;transcription factor E4TF1-53; nuclear respiratory factor-2 subunit beta2 (NRF- 2B) GABPβ2 GABPB1 GA binding protein beta-2 chain Bannert GABPB(GABPB2); GABP-beta-2 sub- 1999 (ibid) E4TF1B unit; transcription factorE4TF1-47 GATA1 GATA1 globin transcription factor 1; Goodman GF1GATA-binding protein 1 2000 (ibid) ERYF1 erythroid transcription factor;NFE1 ERYF1; GF1; NF-E1 Gli3 GLI3 zinc finger protein GLI3; GoodmanPAP-A; GCPS; GLI-Kruppel 2000 (ibid) family member GLI3 (Greigcephalopolysyndactyly syndrome); Pallister-Hall syndrome (PHS) GR NR3C1glucocorticoid receptor (GR); Pfitzner GRL nuclear receptor subfamily 3,1998 (ibid), GCR group C, member 1 (NR3C1); Hottiger GRL 2000 (ibid)HIF1α HIF1A hypoxia-inducible factor-1 α Goodman (HIF1α); ARNTinteracting 2000 (ibid), protein; member of PAS protein Bhattacharya 1;MOP1 1999²⁹, Kallio 1998³⁰, Ema 1999³¹, Hottiger 2000 (ibid) HNF4α HNF4Aheaptocyte nulcear factor-1 α; Goodman NR2A1 HNF-4-α; transcriptionfactor 2000 (ibid), TCF14 HNF-4; transcription factor 14; Soutoglou HNF4MODY; maturity onset diabetes 2000³² of the young 1; MODY1; HNF4A;NR2A1; TCF14; HNF IRF-3 IRF3 interferon regulatory factor-3 Goodman(IRF-3) 2000 (ibid), Yoneyama 1998³³ JunB JUNB transcription factorJunB; proto- Goodman oncogene JunB 2000 (ibid) Mdm2 MDM2 mouse doubleminute 2; human Goodman homolog of p53-binding protein 2000 (ibid)(Mdm2); ubiquitin-protein ligase E3 Mdm2; EC 6.3.2.-; p53- bindingprotein Mdm2; onco- protein Mdm2; double minute 2 protein; Hdm2 MEF2CMEF2C myocyte enhancer factor 2C Sartorelli (MEF2C); myocyte-specificen- 1997 (ibid) hancer factor 2C; MADS box transcription enhancer factor2 polypeptide C Mi MITF microphthalmia-associated trans- Goodmancription factor 2000 (ibid), Sato 1997³⁴ MyoD MYOD1M myoblastdetermination protein Yuan 1996 YF3 1 (MyoD); myogenic factor Ref,MYF-3; myogenic factor 3; Sartorelli PUM 1997³⁵ NF-AT1 NFAT1 nuclearfactor of activated T Garcia- NFATC2 cells, cytoplasmic 2; T cellRodriguez NFATP transcription factor NFAT1; 1998³⁶, Sisk NFATpre-existing subunit; NF- 2000³⁷ ATp NF-YB NFYB NF-Y protein chain B(NF-YB); Li 1998³⁸, HAP3 nuclear transcription factor Y Faniello subunitbeta; α-CP1, CP1; 1999³⁹ CCAAT-binding transcription factor subunit A(CBF-A); CAAT-box DNA binding protein subunit B NF-YA NFYA NF-Y proteinchain A (NF-YA); Li 1998 HAP2 CCAAT-binding transcription (ibid) factorsubunit B (CBF-B); CAAT-box DNA binding protein subunit A; nucleartranscription factor Y α RelA RELA NF-κB RelA, transcription fac-Hottiger NFKB3 tor p65; nuclear factor NF- 1998⁴⁰, kappa-B, p65 subunit;v-rel Gerritsen avian reticuloendotheliosis viral 1997⁴¹, oncogenehomolog A; nuclear Speir factor of kappa light polypeptide 2000⁴², geneenhancer in B-cells 3 (p65) Hottiger 2000 (ibid) P/CAF P/CAFp300/cbp-associated factor Goodman 2000 (ibid) p/CIP TRAM-1 p300/cbpinteracting protein (p/ Goodman NCOA3 CIP); thyroid hormone receptor2000 (ibid) AIB1 activator molecule; DJ1049g16.2; nuclear receptorcoactivator 3 (thyroid hormone receptor activator molecule TRAM-1;receptor-associated coactivator RAC3; amplified in breast cancer AIB1;ACTR PPARγ PPARG peroxisome proliferator activated Iannone NR1C3receptor γ (PPARG); PPAR- 2001⁴³, gamma; PPARG1; PPARG2 Kodera 2000⁴⁴MRG1 CITED2 Cbp/p300-interacting transacti- Bhattacharya MRG1 vator 2;MSG-related protein 1; 1999 (ibid), melanocyte-specific gene 1; Han2001⁴⁵ MRG1 protein p45 NFE2 nuclear factor, erythroid-derived GoodmanNF-E2 2 45 kDa subunit; NF-E2 45 2000 (ibid) kDa subunit (p45 NF-E2);leucine zipper protein NF-E2 p53 TP53 cellular tumor antigen p53;Goodman P53 tumor suppressor p53;, phos- 2000 (ibid), phoprotein p53;Li-Fraumeni Avantaggiati syndrome 1997⁴⁶ Van Order 1999⁴⁷, Hottiger 2000(ibid) p73 TP73 tumor protein p73; p53-like Goodman P73 transcriptionfactor; p53-related 2000 (ibid) protein Pit-1 POU1F1 pituitary-specificpositive trans- Goodman PIT1 cription factor 1; PIT-1; growth 2000(ibid) GHF1 hormone factor 1, GHF-1; POU domain, class 1, transcriptionfactor 1 RSK1 RPS6KA1 90-kDA ribosomal S6 kinase, Goodman RSK1 ribosomalprotein S6 kinase 2000 (ibid), alpha 1; EC 2.7.1.-; S6K-alpha Hottiger1; 90 kDa ribosomal protein S6 2000 (ibid) kinase 1; p90-RSK1;,ribosomal S6 kinase 1; RSK-1; pp90RSK1; HU-1 RSK3 RPS6KA2 Ribosomalprotein S6 kinase Hottiger RSK3 alpha 2; EC 2.7.1.-; S6K-alpha 2000(ibid) 2; 90 kDa ribosomal protein S6 kinase 2;, p90-RSK 2; ribosomal S6kinase 3; RSK-3; pp90RSK3; HU-2 RSK2 RPS6KA3 ribosomal protein S6 kinaseHottiger RSK2 alpha 3; EC 2.7.1.-; S6K-alpha 2000 (ibid) ISPK1 3; 90 kDaribosomal protein S6 kinase 3; p90-RSK 3; ribosomal S6 kinase 2; RSK-2;pp90RSK2; Insulin-stimulated protein kinase 1; ISPK-1; HU-2;, HU-3 RARγRARG retinoic acid receptor γ (RARγ); Hottiger NR1B3 retinoic acidreceptor gamma-1, 2000 (ibid), RAR-gamma-1; RARC; retinoic Yang 2001⁴⁸acid receptor gamma-2; RAR- gamma-2 RNA DDX9 ATP-dependent RNA helicaseGoodman helicase NDH2 A; nuclear DNA helicase II 2000 (ibid) A (NDH II);DEAD-box protein 9; leukophysin (LKP) RXRα RXRA retinoic acid receptorRXR-α Goodman NR2B1 2000 (ibid), Yang 2001 (ibid) ELK4 ELK4 ETS-domainprotein ELK-4; Goodman SAP1 serum response factor accessory 2000 (ibid),protein 1 (SAP-1); SRF acces- Hottiger sory protein 1 2000 (ibid) SF-1NR5A1 steroidogenic factor 1 (STF-1, Goodman FTZF1 SF-1); steroidhormone receptor 2000 (ibid) AD4BP AD4BP; Fushi tarazu factor SF1(Drosophila) homolog 1; FTZ1; ELP; NR5A1 (nuclear receptor subfamily 5,group A, member 1) Smad3 MADH3 mothers against decapentaplegic GoodmanSMAD3 (Drosophila) homolog 3 (SMAD 2000 (ibid), MAD3 3); mothers againstDPP homo- Janknecht log 3; Mad3; hMAD-3; mMad3; 1998⁴⁹, Feng JV15-2;hSMAD3 1998⁵⁰, Pouponnot 1998 (ibid) Smad4 MADH4 mothers againstdecapentaplegic de Caes- SMAD4 (Drosophila) homolog 4 (SMAD tecker⁵¹,DPC4 4); mothers against DPP homo- Pouponnot log 4; deletion target inpancre- 1998 (ibid) atic carcinoma 4, hSMAD4 Smad1 MADH1 mothers againstdecapentaplegic Pearson SMAD1 (Drosophila) homolog 1 (SMAD 1999⁵², MADR11); mothers against DPP homo- Pouponnot BSP1 log 1; Mad-related protein1; 1998⁵³ transforming growth factor-beta signaling protein-1; BSP-1;hSMAD1; JV4-1 Smad2 MADH2 mothers against decapentaplegic PouponnotSMAD2 (Drosophila) homolog 2 (SMAD 1998 (ibid) MADR2 2); mothers againstDPP homo- log 2; Mad-related protein 2; hMAD-2; JV18-1; hSMAD2 SRC-1SRC1 steroid receptor coactivtor - 1 Goodman NCOA1 (SRC-1); F-SRC-1;nuclear re- 2000 (ibid), ceptor coactivator 1 (NCoA-1); Hottiger SRC12000 (ibid) SREBP1 SREBF1 sterol regulatory element binding GoodmanSREBP1 protein-1 (SREBP-1); sterol 2000 (ibid), regulatoryelement-binding Oliner transcription factor 1 1996⁵⁴ SREBP2 SREBF2sterol regulatory element binding Goodman SREBP2 protein-2 (SREBP-2);sterol 2000 (ibid), regulatory element-binding Oliner transcriptionfactor 2 1996 (ibid) Stat-1 STAT1 signal transducer and activatorGoodman or transcription - 1 α/β; trans- 2000 (ibid), cription factorISGF-3 com- Paulson ponents p91/p84; signal trans- 1999⁵⁵, ducer andactivator of trans- Hottiger cription 1, 91 kD (STAT91) 1998 (ibid),Gingras 1999 (ibid), Zhang 1996⁵⁶ Stat-2 STAT2 signal transducer andactivator Goodman or transcription - 2 (STAT2);; 2000 (ibid), signaltransducer and activator Paulson of transcription 2, 113 kD 1999 (ibid),(STAT113); p113 Hottiger 1998 (ibid), Gringras 1999 (ibid), Bhattacharya1996⁵⁷, Hottiger 2000 (ibid) Stat-3 STAT3 signal transducer andactivator Paulson APRF or transcription - 3; acute-phase 1999 (ibid),response factor Hottiger 1998 (ibid) Stat-4 STAT4 signal transducer andactivator Paulson or transcription - 4 1999 (ibid) Stat-5 STAT5 signaltransducer and activator Paulson STAT5A or transcription - 5A (STAT5A);1999 (ibid) STAT5B MGF; signal transducer and acti- check, vator ortranscription - 5B Gingras (STAT5B); STAT5 1999 (ibid), Pfitzner 1998⁵⁸Stat-6 STAT6 signal transducer and activator Paulson or transcription -6 (STAT6); 1999 (ibid) IL-4 Stat; D12S1644 check, Gingras 1999⁵⁹ TAL1TAL1 T-cell acute lymphocytic leu- Goodman SCL kemia-1 protein; TAL-1protein; 2000 (ibid) TCL5 STEM cell protein; T-cell leu-kemia/lymphoma-5 protein TBP TBP TATA box binding protein Goodman TFIID(TBP); transcription initiation 2000 (ibid) TF2D factor TFIID; TATA-boxfactor; TATA sequence-binding protein; SCA17; GTF2D1; HGNC: 15735; GTF2DTFIIB TFIIB transcription factor IIB (TFIIB, Goodman TF2B TF2B);transcription initiation 2000 (ibid), GTF2B factor IIB; generaltranscription Hottiger factor IIB (GTFIIB, GTF2B) 2000 (ibid) THRA THRAthyroid hormone receptor α Hottiger NR1A1 (THRA); C-erbA-alpha; c-erbA-2000 (ibid) THRA1 1; EAR-7; EAR7; AR7; avian ERBA1 erythroblasticleukemia viral (v-erb-a) oncogene homolog; ERBA; THRA1; THRA2; THRA3;EAR-7.1/EAR-7.2 THRB THRB thyroid hormone receptor β1 Hottiger NR1A2(THRB); thyroid hormone re- 2000 (ibid) THR1 ceptor, beta; avianerythroblastic ERBA2 leukemia viral (v-erb-a) onco- gene homolog 2;THRB1; THRB2; ERBA2; NR1A2; thyroid hormone receptor β2 (THRB) TwistTWIST Twist related protein; H-twist; Goodman acrocephalosyndactyly 32000 (ibid), (Saethre-Chotzen syndrome); Hamamori twist (Drosophila)homolog; 1999⁶⁰ acrocephalosyndactyly 3 (ACS3) YY1 YY1 Ying Yang 1(YY1); transcrip- Goodman tional repressor protein YY1; 2000 (ibid)delta transcription factor; NF- E1; UCRBP; CF1; Yin Yang 1; DELTA; YY1transcription factor Viral E1A Goodman 2000 (ibid), Hottiger 2000 (ibid)EBNA2 EBV Goodman 2000 (ibid) Py LT polyomavirus large T antigen Goodman2000 (ibid) SV40 LT simian virus 40 large T antigen, Goodman TAg 2000(ibid), Hottiger 2000 (ibid) HPV E2 human papillomavirus E2 Goodman 2000(ibid) HPV E6 human papillomavirus E6 Goodman 2000 (ibid), Hottiger 2000(ibid) Tat HIV-1 Goodman 2000 (ibid), Hottiger 2000 (ibid) Tax HumanT-cell leukemia virus Goodman type 1 2000 (ibid), Hottiger 2000 (ibid)Bacterial JMY H pylori Goodman (cag) 2000 (ibid)

[0284] The two major lists are from reviews by Goodman 2000⁶¹ andHottiger 2000⁶².

[0285] Mutations in some of these p300 factors are currently associatedwith chronic diseases. HNF4A with MODY, ESR1 with breast cancer andbronchial asthma, GR with cortisol resistance, etc. Consider thefollowing definition.

[0286] (4) p300/cbp Regulated (Gene, Polypeptide)

[0287] Definition

[0288] Assume the gene G is transactivated, or suppressed by thetranscription complex C. If C contains p300/cbp, the gene G, and thepolypeptide encoded by G, are called “p300/cbp regulated.”

[0289] Exemplary Assays

[0290] 1. Co-transfect a cell with the gene promoter fused to a reportergene, such as CAT or LUC, and a vector expressing p300/cbp. Assayreporter gene expression in the p300/cbp transfected cell and in controlcells transfected with the fused gene promoter along with an “empty”plasmid. If reporter gene expression is higher or lower in the p300/cbptransfected cell, the gene is p300/cbp regulated.

[0291] 2. Select a cell which expresses the gene of interest andtransfect it with a vector expressing p300/cbp. Assay endogenous geneexpression in the p300/cbp transfected cell and in control cellstransfected with an “empty” plasmid. If gene expression is higher orlower in the p300/cbp transfected cell, the gene is p300/cbp regulated.

[0292] Note:

[0293] Preferably, verify that co-transfection did not induce a changein cellular microcompetition, a mutation in the gene promoter, or achange in methylation of gene promoter.

[0294] 3. Transfect a cell with the gene promoter fused to a reportergene, such as CAT or LUC. Contact the cell with an antibody againstp300/cbp (or with a protein such as E1A). Assay gene expression in theantibody treated cell and in the untreated controls. If reporter geneexpression is higher or lower in the antibody treated cell, the gene isp300/cbp regulated.

[0295] 4. Select a cell which expresses a gene of interest. Contact thecell with an antibody against p300/cbp (or with a protein such as E1A).Assay gene expression in both the treated cell and in the untreatedcontrols. If gene expression is higher or lower in the antibody treatedcell, the gene is p300/cbp regulated.

[0296] 5. Perform chromatin assembly of the gene promoter, for instance,with chromatin assembly extract from Drosophila embryos. Add atranscription factor during the chromatin assembly reactions. After thechromatin assembly reaction is complete add the p300/cbp proteins. Allowtime for the interaction of the proteins with the chromatin template.Perform in vitro transcription reaction. Measure the concentration ofthe RNA products, by for instance, primer extension analysis. Compare tothe RNA products before the addition of the p300/cbp proteins. If theaddition of p300/cbp increased the concentration of the RNA products,the gene is p300/cbp regulated.

[0297] 6. See more assays below.

[0298] Examples

[0299] Direct evidence shows transactivation of certain promoters byp300/cbp (Manning 2001⁶³, Kraus 199⁶⁴, Kraus 1998⁶⁵).

[0300] Indirect evidence is available in studies with p300/cbp factors.Consider, for example, the p300/cbp factor GABP. GABP binds promotersand enhancers of many cellular genes including β₂ leukocyte integrin(CD18) (Rosmarin 1998⁶⁶), interleukin 16 (IL-16) (Bannert 1999⁶⁷),interleukin 2 (IL-2) (Avots 1997⁶⁸), interleukin 2 receptor β-chain(IL-2Rβ) (Lin 1993⁶⁹), IL-2 receptor γ-chain (IL-2 γc) (Markiewicz1996⁷⁰), human secretory interleukin-1 receptor antagonist (secretoryIL-1ra) (Smith 1998⁷¹), retinoblastoma (Rb) (Sowa 1997⁷²), humanthrombopoietin (TPO) (Kamura 1997⁷³), aldose reductase (Wang 1993⁷⁴),neutrophil elastase (NE) (Nuchprayoon 1999⁷⁵, Nuchprayoon 1997⁷⁶),folate binding protein (FBP) (Sadasivan 1994⁷⁷), cytochrome c oxidasesubunit Vb (COXVb) (Basu 1993⁷⁸, Sucharov 1995⁷⁹), cytochrome c oxidasesubunit IV (Carter 1994⁸⁰, Carter 1992⁸¹), mitochondrial transcriptionfactor A (mtTFA) (Virbasius 1994⁸²), β subunit of the FoF1 ATP synthase(ATPsynβ) (Villena 1998⁸³), prolactin (prl) (Ouyang 1996⁸⁴) and theoxytocin receptor (OTR) (Hoare 1999⁸⁵) among others. For some of thesegenes, for instance, CD18, COXVb, COXIV, GABP binds to the promoterwhile for others, for example IL-2 and ATPsynβ, GABP binds an enhancer.More examples see below.

[0301] Another p300/cbp factor is NF-Y (see above). Mantovani 1998⁸⁶,provides a list of genes which include a NF-Y binding site (Mantovani1998, ibid, Table 1). For the listed genes, the table indicates whetherthe referenced studies report the presence of a proven binding site fora transcription factor close to the NF-Y binding site, whethercross-competition data with bonafide NF-Y binding sites are available,whether EMSA supershift experiments with anti NF-Y antibodies wereperformed, and whether the studies performed in vitro or in vivotransactivation studies with NF-Y. Some of the genes listed in the paperare MCH II, Ii, Mig, GP91 Phox, CD10, RAG-1, IL4, Thy-1, globin α,ζ,γ^(D) γ^(P), Coll α2 (I) α1 (I), osteopontin, BSP, apoA-I, aldolase B,TAT, γ-GT, SDH, fibronectin, arg lyase, factor VIII, factor X, MSP,ALDH, LPL, ExoKII, FAS, TSP-1, FGF-4, α1-chim, Tr Hydr, NaKATPsea-3,PDFGβ, FerH, MHC IA2 B8, Cw2Ld and B7, MDR1, CYP1A1, c-JUN, Grp78,Hsp70, ADH2, GPAT, FPP, HMG, HSS, SREBP2, GHR, CP2, β-actin, TK,TopoIIα, I, II, III, IV, cdc25, cdc2, cyc1A, cyc1B1, E2F1, PLK, RRR2,HisH2B, HisH3.

[0302] (5) p300/cbp Factor Kinase (p300/cbp Factor Phosphatase)

[0303] Definition

[0304] Assume F is a p300/cbp factor. If a molecule L stimulatesphosphorylation or dephosphorylation of F, L is called “p300/cbp factorkinase” or “p300/cbp factor phosphatase”, respectively.

[0305] Exemplary Assays

[0306] 1. Contact a system (for instance, organism, cell, cell lysate,chemical mixture) with a test molecule L. Use assays described in thesection entitled “Assaying protein phosphorylation,” or similar assays,to uncover a change in phosphorylation of the p300/cbp factor ofinterest. An increase in phosphorylation indicates that L is a p300/cbpfactor kinase, and a decrease indicates that L is a p300/cbp factorphosphatase.

[0307] Example

[0308] Ras, Raf, MEK1, MEK 2, MEK4, ERK, JNK, three classes of ERKinactivators: type 1/2 serine/threonine phosphatases, such as PP2A,tyrosine-specific phosphatases (also called protein-tyrosinephosphatase, denoted PTP), such as PTP1B, and dual specificityphosphatases, such as MKP-1 which affect phosphorylation of a number oftranscription factors, for instance, GABP, NF-κB. See also below.

[0309] (6) p300/cbp Agent

[0310] Definition

[0311] Assume the polynucleotide Pn binds the transcription complex C.Assume C contains p300/cbp. If a molecule L stimulates or suppressesbinding of C to Pn, L is called “p300/cbp agent.” Specifically, such anagent can stimulate or suppress binding of p300/cbp to a p300/cbpfactor, binding of p300/cbp to DNA, or binding of a p300/cbp factor toDNA.

[0312] Exemplary Assays

[0313] 1. Contact a system (for instance, whole organism, cell, celllysate, chemical mixture) with a test molecule L. Use assays describedin the section entitled “Assaying binding to DNA,” or similar assays, touncover a change in binding of the C to DNA. Specifically, assay forbinding between p300/cbp and DNA, or p300/cbp and a p300/cbp factor, orp300/cbp factor and DNA.

[0314] Examples

[0315] Examples of p300/cbp agents include sodium butyrate (SB),trichostatin A (TSA), trapoxin (for SB, TSA and trapoxin see in Espinos1999⁸⁷), phorbol ester (phorbol 12-myristate 13-acetate, PMA, TPA),thapsigargin (for PMA and thapsigargin see Shiraishi 2000⁸⁸, for PMA seeHerrera 1998⁸⁹, Stadheim 1998⁹⁰), retinoic acid (RA, vitamin A) (Yen1999⁹¹), interferon-γ (IFNγ) (Liu 1994⁹², Nishiya 1997⁹³), heregulin(HRG, new differentiation factor, NDF, neuregulin, NRG) (Lessor 1998⁹⁴,Marte 1995⁹⁵, Sepp-Lorenzino 1996⁹⁶, Fiddes 1998⁹⁷), zinc (Zn) (Park1999⁹⁸, Kiss 1997⁹⁹), copper (Cu) (Wu 1999¹⁰⁰, Samet 1998¹⁰¹, bothstudies also show phosphorylation of ERK1/2 by Zn), estron, estradiol(Migliaccio 1996¹⁰², Ruzycky 1996¹⁰³, Nuedling 1999¹⁰⁴), interleukin 1β(IL-1β) (Laporte 1999¹⁰⁵, Larsen 1998¹⁰⁶), interleukin 6 (IL-6)(Daeipoou 1993¹⁰⁷), tumor necrosis factor α (TNFα) (Leonard 1999¹⁰⁸),transforming growth factor β (TGFβ) (Hartsough 1995¹⁰⁹, Yonekura1999¹¹⁰, oxytocin (OT) (Strakova 1998¹¹¹, Copland 1999¹¹², Hoare1999¹¹³). All studies show phosphorylation of ERK1/2 by these agents.See more agents below.

[0316] Other examples include agents which modify oxidative stress, suchas, diethyl maleate (DEM), a glutathione (GSH)-depleting agent, andN-acetylcysteine (NAC), an antioxidant and a precursor of GSH synthesis.See more agents below.

[0317] (7) Foreign p300/cbp Polynucleotide

[0318] Definition

[0319] Assume Pn is a polynucleotide foreign to organism R. If Pn is ap300/cbp polynucleotide, Pn is called “p300/cbp polynucleotide foreignto R.”

[0320] Exemplary Assays

[0321] Combine assays in the p300/cbp polynucleotide and foreignpolynucleotide sections above.

[0322] Examples

[0323] See examples in “p300/cbp virus” below.

[0324] (8) p300/cbp Virus

[0325] Definition

[0326] Assume Pn is a p300/cbp polynucleotide. If Pn is a segment of thegenome of a virus V, V is called a “p300/cbp virus.”

[0327] Exemplary Assays

[0328] 1. Verify that Pn is a p300/cbp polynucleotide (see assaysabove). Compare the sequence of Pn with the sequence of the published Vgenome. If the sequence is a segment of the V genome, Pn is a p300/cbpvirus. If the V genome is not published, its sequence can be determinedempirically.

[0329] 2. Verify that Pn is a p300/cbp polynucleotide (see assays above)by hybridizing Pn to the V genome. If Pn hybridizes, Pn is a p300/cbpvirus.

[0330] Examples

[0331] Direct evidence shows transactivation of certain viruses byp300/cbp. See, for instance, Subramanian 2002¹¹⁴ on Epstein-Barr virus,Banas 2001, Deng 2000¹¹⁵ on HIV-1¹¹⁶, Cho 2001¹¹⁷ on SV40 andpolyomavirus, Wong 1994¹¹⁸, on adenovirus type 5. See also Hottiger2000¹¹⁹, a review on viral replication and p300/cbp.

[0332] Indirect evidence is available in studies with p300/cbp factors.Consider, for instance, the p300/cbp factor GABP. Since GABP bindsp300/cbp (see above), a complex on DNA which includes GABP, alsoincludes p300/cbp. The DNA motif (A/C)GGA(A/T)(G/A), termed the N-box,is the core binding sequence for GABP. The N-box is the core bindingsequence of many viral enhancers including the polyomavirus enhancerarea 3 (PEA3) (Asano 1990¹²⁰), adenovirus E1A enhancer (Higashino1993¹²¹), Rous Sarcoma Virus (RSV) enhancer (Laimins 1984¹²²), HerpesSimplex Virus 1 (HSV-1) (in the promoter of the immediate early geneICP4) (LaMarco 1989¹²³, Douville 1995¹²⁴), Cytomegalovirus (CMV) (IE-1enhancer/promoter region) (Boshart 1985¹²⁵), Moloney Murine LeukemiaVirus (Mo-MuLV) enhancer (Gunther 1994¹²⁶), Human Immunodeficiency Virus(HIV) (the two NF-κB binding motifs in the HIV LTR) (Flory 1996¹²⁷),Epstein-Barr virus (EBV) (20 copies of the N-box in the +7421/+8042oriP/enhancer) (Rawlins 1985¹²⁸) and Human T-cell lymphotropic virus(HTLV) (8 N-boxes in the enhancer (Mauclere 1995¹²⁹) and one N-box inthe LTR (Kornfeld 1987¹³⁰)). Moreover, some viral enhancers, for exampleSV40, lack a precise N-box, but still bind the GABP transcription factor(Bannert 1999¹³¹).

[0333] Ample evidence exists which supports the binding of GABP to theN-boxes in these viral enhancers. For instance, Flory, et al., 1996¹³²show binding of GABP to the HIV LTR, Douville, et al., 1995¹³³ showbinding of GABP to the promoter of ICP4 of HSV-1, Bruder, et al.,1991¹³⁴ and Bruder, et al., 1989¹³⁵ show binding of GABP to theadenovirus E1A enhancer element I, Ostapchuk, et al., 1986¹³⁶ showbinding of GABP (called EF-1A in this paper) to the polyomavirusenhancer and Gunther, et al., 1994¹³⁷ show binding of GABP to Mo-MuLV.

[0334] Other studies demonstrate competition between these viralenhancers and enhancers of other viruses. Scholer and Gruss, 1984¹³⁸show competition between the Moloney Sarcoma Virus (MSV) enhancer andSV40 enhancer and also competition between the RSV enhancer and the BKvirus enhancer.

[0335] Another p300/cbp factor is NF-Y (see above). Mantovani 1998(ibid), provides a list of viruses which include a NF-Y binding site(Table 1). The list includes HBV S, MSV LTR, RSV LTR, ad EIIL II, Ad MK,CMV gpUL4, HSV IE110k, VZV ORF62, MVM P4.

[0336] More Exemplary Assays for Identification of a Polynucleotide Pnas a p300/cbp Polynucleotide:

[0337] 1. Take a cell of interest. Modify the copy number of Pn in thecell (by, for instance, transfection, infection, mutation, etc, see alsoabove). Assay binding of all p300/cbp factors to Pn. If a p300/cbpfactor binds Pn, Pn is a p300/cbp polynucleotide.

[0338] 2. Assay binding of a p300/cbp factor to endogenous DNA or toexogenous DNA following introduction to the cell of interest. Modify thecopy number of Pn in the cell. Assay binding of the p300/cbp factoragain. If binding changed, Pn is a p300/cbp polynucleotide.

[0339] 3. Identify a binding site on Pn for p300/cbp or a p300/cbpfactor by computerized sequence analysis.

[0340] 4. Take a cell of interest. Co-transfect a vector expressing Pn(or change the copy number of Pn in the cell through other means), and apromoter of a p300/cbp regulated gene fused to a reporter gene. Assayreporter gene expression and compare to cells co-transfected with anempty plasmid. If expression in the Pn transfected cell is differentthan controls, Pn is a p300/cbp polynucleotide.

[0341] 5. Take a cell of interest which express a p300/cbp regulatedgene. Modify the copy number of Pn in the cell (by, for instance,transfection, infection, mutation, etc, see also above). Assayexpression of the p300/cbp regulated gene and compare to cells with anunmodified copy number of Pn (for instance, in cells transfected with anempty plasmid). If expression in the Pn transfected cell is differentthan controls, Pn is a p300/cbp polynucleotide.

[0342] 6. Take a cell of interest. Infect the cell with a p300/cbpvirus. Modify the copy number of Pn in the cell (by, for instance,transfection, infection, mutation, etc, see also above). Assay viralreplication and compare to cells with unmodified copy number of Pn (forinstance, in cells infected with a non p300/cbp virus). If viralreplication is different, Pn is a p300/cbp polynucleotide.

[0343] 7. Compare the sequence of Pn to the genome of a p300/cbp virususing a sequence alignment algorithm such as BLAST. If a segment of thePn sequence is identical (or homologous) to a segment in viral genome,Pn is a p300/cbp polynucleotide. A polynucleotide of at least 18nucleotides should be sufficient to ensure specificity and validatealignment.

[0344] 8. Try to hybridize Pn to the genome of a p300/cbp virus. If Pnhybridizes to the viral genome, Pn is a p300/cbp polynucleotide.Hybridization conditions should be sufficiently stringent to permitspecific, but not promiscuous, hybridization. Such conditions are wellknown in the art.

c) Agents Related Elements

[0345] (1) Modulator

[0346] Definition

[0347] Consider a polynucleotide Pn. An agent, or treatment (calledagent for short), is called “modulator” if the agent modifiesmicrocompetition with Pn, modifies at least one effect ofmicrocompetition with Pn, or modifies at least one effect of anotherforeign polynucleotide-type disruption.

[0348] Notes:

[0349] 1. A treatment, such as irradiation, can also be a modulator. Inprinciple, according to the definition, any foreign polynucleotide-typedisruption is a modulator.

[0350] Exemplary Assays

[0351] 1. Assay the effect of an agent on Pn copy number.

[0352] Specifically, take a biological system (e.g. cell, wholeorganism, etc). Modify the copy number of Pn (by, for instance,transfection, infection, mutation, etc, see above). Call this cell thePn cell. Assay the Pn copy number in the Pn cell (see above). Contactthe biological system with an agent of interest. Assay again the Pn copynumber. If the Pn copy number is higher or lower compared to the copynumber in Pn cells not contacted with the agent, the agent is amodulator.

[0353] 2. Assay the effect of an agent on binding of p300/cbp to Pn,directly or in a complex.

[0354] Specifically, take a biological system (e.g. cell, wholeorganism, etc). Modify the copy number of Pn (by, for instance,transfection, infection, mutation, etc, see above). Call this cell thePn cell. Assay binding of p300/cbp to Pn (see above). Contact thebiological system with an agent of interest. Assay again the binding ofp300/cbp to Pn. If the binding is higher or lower compared to binding inPn cells not contacted with the agent, the agent is a modulator.

[0355] 3. Assay the effect of an agent on binding of a p300/cbp factorto Pn.

[0356] Specifically, take a biological system (e.g. cell, wholeorganism, etc). Modify the copy number of Pn (by, for instance,transfection, infection, mutation, etc, see above). Call this cell thePn cell. Assay binding of a p300/cbp factor to Pn (see above). Contactthe biological system with an agent of interest. Assay again the bindingof the p300/cbp factor to Pn. If binding is higher or lower compared tobinding in Pn cells not contacted with the agent, the agent is amodulator.

[0357] 4. Assay the effect of an agent on binding of p300/cbp to ap300/cbp factor.

[0358] Specifically, take a biological system (e.g. cell, wholeorganism, etc). Modify the copy number of Pn (by, for instance,transfection, infection, mutation, etc, see above). Call this cell thePn cell. Assay binding of p300/cbp to a p300/cbp factor (see above).Contact the biological system with an agent of interest. Assay again thebinding of p300/cbp to a p300/cbp factor. If binding is higher or lowercompared to binding in Pn cells not contacted with the agent, the agentis a modulator.

[0359] 5. Assay the effect of an agent on expression of a disrupted geneand/or polypeptide.

[0360] Specifically, take a biological system (e.g. cell, wholeorganism, etc). Modify the copy number of Pn (by, for instance,transfection, infection, mutation, etc, see above). Call this cell thePn cell. Identify a disrupted gene and/or polypeptide (see assaysabove). Contact the biological system with an agent of interest. Assaythe bioactivity of the disrupted gene and/or polypeptide. If thebioactivity of the disrupted gene and/or polypeptide is higher or lowercompared to the bioactivity in Pn cells not contacted with the agent,the agent is a modulator.

[0361] Examples

[0362] See below in constructive/disruptive.

[0363] (2) Constructive/Disruptive

[0364] Definition

[0365] A modulator, which attenuates or accentuates microcompetitionwith a foreign polynucleotide, attenuates or accentuates at least oneeffect of microcompetition with a foreign polynucleotide, or attenuatesor accentuates at least one effect of another foreignpolynucleotide-type disruption, is called “constructive” or“disruptive,” respectively.

[0366] Notes:

[0367] 1. A modulator can be both constructive and disruptive.

[0368] 2. Consider a gene suppressed by microcompetition with a foreignpolynucleotide. Consider such a gene in a cell without a foreignpolynucleotide. Now consider a mutation which reduces the genebioactivity. An agent which stimulates expression of such mutated genewill also be called constructive. If, on the other hand, the mutationstimulates the gene bioactivity, an agent which suppresses itsbioactivity will also be called constructive.

[0369] 3. A constructive agent can be an agonist, if it stimulatesexpression of a gene suppressed by microcompetition with a foreignpolynucleotide, or if is stimulates bioactivity of a polypeptide encodedby such a gene. A constructive agent can also be an antagonist if itinhibits expression of a gene stimulated by microcompetition with aforeign polynucleotide, or inhibits the bioactivity of a polypeptideencoded by such a gene.

[0370] 4. A foreign polynucleotide-type disruption can be constructive.

[0371] Exemplary Assays

[0372] 1. See assays in Modulator section above. In these assay ifeither;

[0373] (a) Pn copy number in the Pn cell contacted with the agent ishigher relative to Pn cells not contacted by the agent;

[0374] (b) binding of p300/cbp to Pn in the Pn cell contacted with theagent is higher compared to binding in Pn cells not contacted with theagent;

[0375] (c) binding of p300/cbp factor to Pn in the Pn cell contactedwith the agent is higher compared to binding in Pn cells not contactedwith the agent;

[0376] (d) binding of p300/cbp to a p300/cbp factor in the Pn cellcontacted with the agent is higher or lower compared to binding in Pncells not contacted with the agent;

[0377] (e) bioactivity of the disrupted gene and/or polypeptide in thePn cell contacted with the agent is higher (for genes and/orpolypeptides with suppressed bioactivity) compared to the bioactivity inPn cells not contacted with the agent;

[0378] the agent is constructive.

[0379] If the effect is in the opposite direction, the agent isdisruptive.

[0380] Examples

[0381] Antiviral drugs, sodium butyrate, garlic, etc. See more examplesin Treatment section below.

2. Detailed Description of Standard Elements a) General Comments

[0382] The elements of the present invention may include, as their ownelements, standard methods in molecular biology, microbiology, cellbiology, transgenic biology, recombinant DNA, immunology, cell culture,pharmacology, and toxicology, well known in the art. The followingsections provide details for some standard methods. Completedescriptions are available in the literature. For instance, see the“Current Protocols” series published by John Wiley & Sons. The followinglist provides a sample of books in the series: Current Protocols in CellBiology, edited by: Juan S. Bonifacino, Mary Dasso, JenniferLippincott-Schwartz, Joe B Harford, and Kenneth M Yamada; CurrentProtocols in Human Genetics, edited by: Nicholas C Dracopoli, Jonathan LHaines, Bruce R Korf, Cynthia C Morton, Christine E Seidman, J GSeidman, Douglas R Smith; Current Protocols in Immunology, edited by:John E Coligan, Ada M Kruisbeek, David H Margulies, Ethan M Shevach, andWarren Strober; Current Protocols in Molecular Biology, edited by:Frederick M Ausubel, Roger Brent, Robert E Kingston, David D Moore, J GSeidman, John A Smith, and Kevin Struhl; Current Protocols in NucleicAcid Chemistry, edited by: Serge L Beaucage, Donald E Bergstrom, Gary DGlick, Roger A Jones; Current Protocols in Pharmacology, edited by: S JEnna, Michael Williams, John W Ferkany, Terry Kenakin, Roger D Porsolt,James P Sullivan; Current Protocols in Protein Science, edited by: JohnE Coligan, Ben M Dunn, Hidde L Ploegh, David W Speicher, Paul TWingfield; Current Protocols in Toxicology, edited by: Mahin Maines(Editor-in-Chief), Lucio G Costa, Donald J Reed, Shigeru Sassa, I GlennSipes. The following lists includes more books with standard methods.Basic DNA and RNA Protocols (Methods in Molecular Biology, Vol 58),edited by Adrian J Harwood, Humana Press, 1994, DNA-ProteinInteractions: Principles and Protocols (Methods in Molecular Biology,Volume 148), edited by Tom Moss, Humana Press, 2001, TranscriptionFactor Protocols (Methods in Molecular Biology), edited by Martin JTymms, Humana Press, 2000, Gene Transcription: A Practical Approach,edited by B D Hames, and S J Higgins, IRL Press at Oxford UniversityPress, 1993, Gene Transcription, DNA Binding Proteins: EssentialTechniques, edited by Kevin Docherty, Jossey Bass, 1997, Gene ProbesPrinciples and Protocols (Methods in Molecular Biology, 179), edited byMarilena Aquino de Muro and Ralph Rapley, Humana Press, 2001, GeneIsolation and Mapping Protocols (Methods in Molecular Biology Vol 68),edited by Jackie Boultwood and Jacqueline Boultwood, Humana Press, 1997,Gene Targeting Protocols (Methods in Molecular Biology, Vol 133), editedby Eric B Kmiec and Dieter C Gruenert, Humana Press 2000, EpitopeMapping Protocols (Methods in Molecular Biology, Vol 66), edited byGlenn E Morris, Humana Press, 1996, Protein Targeting Protocols (Methodsin Molecular Biology, Vol 88), edited by Roger A Clegg, Humana Press,1998, Monoclonal Antibody Protocols (Methods in Molecular Biology, 45),edited by William C Davis, Humana Press, 1995, Immunochemical Protocols(Methods in Molecular Biology Vol 80), edited by John D Pound, HumanaPress, 1998, Immunoassay Methods and Protocols (Methods in MolecularBiology), edited by Andrey L Ghindilis, Andrey R Pavlov and Plamen BAtanassov, Humana Press, 2002, In situ Hybridization Protocols (Methodsin Molecular Biology, 123), edited by Ian A Darby, Humana Presse, 2000,Bioluminescence Methods & Protocols, edited by Robert A Larossa, HumanaPress, 1998, Affinity Chromatography: Methods and Protocols (Methods inMolecular Biology), etided by Pascal Bailon, George K Ehrlich, Wen-JianFung, wo Berthold and Wolfgang Berthold, Humana Press, 2000, Protocolsfor Oligonucleotide Conjugates: Synthesis and Analytical Techniques(Methods in Molecular Biology, Vol 26), edited by Sudhir Agrawal, HumanaPress, 1993, RNA Isolation and Characterization Protocols (Methods inMolecular Biology, No 86), edited by Ralph Rapley and David L Manning,Humana Press, 1998, Protocols for Oligonucleotides and Analogs:Synthesis and Properties (Methods in Molecular Biology, 20), edited bySudhir Agrawal, Humana Press, 1993, Basic Cell Culture Protocols(Methods in Molecular Biology, 75), edited by Jeffrey W Pollard and JohnM Walker, Humana Press, 1997, Quantitative PCR Protocols (Methods inMolecular Medicine, 26), edited by Bernd Kochanowski and Udo Reischl,Humana Press, 1999, In situ PCR Techniques, edited by Omar Bagasra andJohn Hansen, John Wiley & Sons, 1997, PCR Cloning Protocols: FromMolecular Cloning to Genetic Engineering (Methods in Molecular Biology,No 67), edited by Bruce A White, Humana Press, 1996, PRINS and In situPCR Protocols (Methods in Molecular Biology, 71), edited by John RGosden, Humana Press, 1996, PCR Protocols: Current Methods andApplications (Methods in Molecular Biology, 15), edited by Bruce AWhite, Humana Press 1993, Transmembrane Signaling Protocols (Methods inMolecular Biology, Vol 84), edited by Daffia Bar-Sagi, Humana Press,1998, Chemokine Protocols (Methods in Molecular Biology, 138), edited byAmanda E I Proudfoot, Timothy N C Wells and Chris Power, Humana Press,2000, Baculovirus Expression Protocols (Methods in Molecular Biology,Vol 39), edited by Christopher D Richardson, Humana Press, 1998,Recombinant Gene Expression Protocols (Methods in Molecular Biology,62), edited by Rocky S Tuan, Humana Press, 1997, Recombinant ProteinProtocols: Detection and Isolation (Methods in Molecular Biology, Vol63), edited by Rocky S Tuan, Humana Press, 1997, DNA Repair Protocols:Eukaryotic Systems (Methods in Molecular Biology, Vol 113), edited byDaryl S Henderson, Humana Press, 1999, DNA Sequencing Protocols, editorsHugh G Griffin and Annette M Griffin, Humana Press, 1993, ProteinSequencing Protocols (Methods in Molecular Biology, No 64), edited byBryan John Smith, Humana Press, 2001, Gene Transfer and ExpressionProtocols (Methods in Molecular Biology, Vol 7), edited by E J Murray,Humana Press, 1991, Transgenesis Techniques, Principles and Protocols(Methods in Molecular Biology, 180), edited by Alan R Clarke, HumanaPress, 2002, Regulatory Protein Modification Techniques and Protocols(Neuromethods, 30), edited by Hugh C Hemmings, Humana Press, 1996,Downstream Processing of Proteins Methods and Protocols (Methods inBiotechnology, 9), edited by Mohamed A Desai, Humana Press, 2000, DNAVaccines Methods and Protocols (Methods in Molecular Medicine, 29),edited by Douglas B Lowrie and Robert Whalen, Humana Press, 1999, DNAArrays Methods and Protocols (Methods in Molecular Biology, 170), editedby Jang B Rampal, Humana Press, 2001, Drug-DNA Interaction Protocols,editor Keith Fox, Humana Press, 1997, In vitro Mutagenesis Protocols,edited Michael K. Trower, Humana Press, 1996, In vitro Toxicity TestingProtocols (Methods in Molecular Medicine, 43), edited by Sheila O'Hareand C K Atterwill, Humana Press, 1995, Mutation Detection: A PracticalApproach (Practical Approach Series (Paper), No 188), edited by RichardG H Cotton, E Edkins and S Forrect, Irl Press, 1998, Herpes SimplexVirus Protocols (Methods in Molecular Medicine, 10), edited by S MoiraBrown and Alasdair R MacLean, Humana Press, 1997, HIV Protocols (Methodsin Molecular Medicine, 17), edited by Nelson Michael and Jerome H Kim,Humana Press, 1999, Cytomegalovirus Protocols (Methods in MolecularMedicine, 33), edited by John Sinclair, Humana Press, 1999, AntiviralMethods and Protocols (Methods in Molecular Medicine, 24), edited byDerek Kinchington and Raymond F Schinazi, Humana Press, 1999,Epstein-Barr Virus Protocols (Methods in Molecular Biology Vol 174),edited by Joanna B Wilson and Gerhard H W May, Humana Press, 2001,Adenovirus Methods and Protocols (Methods in Molecular Medicine, Vol21), edited by William S M Wold, Humana Press, 1999, Molecular Methodsfor Virus Detection, edited by Danny L Wiedbrauk and Daniel H Farkas,Academic Press, 1995, Diagnostic Virology Protocols (Methods inMolecular Medicine, No 12), edited by John R Stephenson and Alan Wames,Humana Press, 1998. A more extensive list of books with detaileddescription of standard methods is available at the Promega web site:

[0383]http://www.promega.com/catalog/category.asp?catalog%5Fname=Promega%5FProducts&category%5Fname=Books&description%5Ftext=Books&Page=1. The Promega listincludes 260 books.

[0384] For each element, one or more exemplary protocols are presented.All examples included in the application should be considered asillustrations, and, therefore, should not be construed as limiting theinvention in any way.

[0385] More details regarding the presented exemplary protocols, anddetails of other protocols that can be used instead of the presentedprotocols, are available in the cited references, and in the bookslisted above. The contents of all references cited in the application,including, but not limited to, abstracts, papers, books, publishedpatent applications, issued patents, available in paper format orelectronically, are hereby expressly and entirely incorporated byreference.

[0386] The following sections first present protocols for formulation ofa drug candidate, then protocols, that as elements of above assays, canbe used to test a drug candidate for a desired biological activityduring drug discovery, development and clinical trials. The assays canalso be used for diagnostic purposes. Finally, the following sectionsalso present protocols for effective use of a drug as treatment.

b) Formulation Protocols

[0387] One aspect of the invention pertains to administration of amolecule of interest, equivalent molecules, or homologous molecules,isolated from, or substantially free of contaminating molecules, astreatment of a chronic disease.

[0388] (1) Definitions

[0389] (a) Molecule of Interest

[0390] The terms “molecule of interest” or “agent,” is understood toinclude small molecules, polypeptides, polynucleotides and antibodies,in a form of a pharmaceutical or nutraceutical.

[0391] (b) Equivalent Molecules

[0392] The term “equivalent molecules” is understood to includemolecules having the same or similar activity as the molecule ofinterest, including, but not limited to, biological activity, chemicalactivity, pharmacological activity, and therapeutic activity, in vitroor in vivo.

[0393] (c) Homologous Molecules

[0394] The term “homologous molecules” is understood to includemolecules with the same or similar chemical structure as the molecule ofinterest.

[0395] In one exemplary embodiment, homologous molecules may besynthesized by chemical modification of a molecule of interest, forinstance, by adding any of a number of chemical groups, including butnot limited to, sugars (i.e. glycosylation), phosphates, acetyls,methyls, and lipids. Such derivatives may be derived by the covalentlinkage of these or other groups to sites within a molecule of interest,or in the case of polypeptides, to the N-, or C-termini, orpolynucleotides, to the 5′ or 3′ ends.

[0396] In one exemplary embodiment, homologous polypeptides orhomologous polynucleotides include polypeptides or polynucleotides thatdiffer by one or more amino acid, or nucleotides, respectively, from thepolypeptide or polynucleotide of interest. The differences may arisefrom substitutions, deletions or insertions into the initial sequence,naturally occurring or artificially formulated, in vivo or in vitro.Techniques well known in the art may be applied to introduce mutations,such as point mutations, insertions or deletion, or introduction ofpremature translational stops, leading to the synthesis of truncatedpolypeptides. In every case, homologs may show attenuated activitiescompared to the original molecules, exaggerated activities, or mayexpress a subset or superset of the total activities elicited by theoriginal molecule. In these ways, homologs of constructive or disruptivepolypeptides or polynucleotides have biological activities eitherdiminished or expanded compared to the original molecule. In every case,a homolog may, or may not prove more effective in achieving a desiredtherapeutic effect. Methods for identifying homologous polypeptides orpolynucleotides are well known in the art, for instance, molecularhybridization techniques, including, but not limited to, Northern andSouthern blot analysis, performed under variable conditions oftemperature and salt, can formulate nucleic acid sequences withdifferent levels of stringency. Suitable protocols for identifyinghomologous polypeptides or polynucleotides are well known in the art(see, for instance, Sambrook 2001¹³⁹ and above listed books of standardprotocols). Homologous polypeptides or polynucleotides can also begenerated, for instance by a suitable combinatorial approach.

[0397] It is well known in the art that the ribonucleotide triplets,termed codons, encoding each amino acid, comprise a set of similarsequences typically differing in their third position. Variations, knownas degeneracy, occur naturally, and in practice mean that any givenamino acid may be encoded by more than one codon. For instance, theamino acids arginine, serine and leucine can be encoded by 6 codons. Asa result, in one exemplary embodiment, homologous DNA and RNApolynucleotides can be produced which encode the same polypeptide ofinterest.

[0398] In another exemplary embodiment, a set of homologous polypeptidesmay be generated by incorporating a population of syntheticoligodeoxyribonucleotides into expression vectors already carryingadditional portions of the polypeptide of interest. The site into whichthe oligonucleotide-gene fusion is incorporated must include appropriatetranscriptional and translational regulatory sequences flanking theinserted oligonucleotides to permit expression in host cells. Onceintroduced into an appropriate host cell, the resulting collection ofgene-oligonucleotide recombinant vectors expresses polypeptide variantsof the polypeptide of interest. The expressed polypeptide may beseparately purified by cloning the vector bearing host cells, or byemploying appropriate bacteriophage vectors, such as gt-11 or itsderivatives, and screening plaques with antibodies against thepolypeptide of interest, or against an immunological tag included in therecombinants.

[0399] (d) Isolated

[0400] The terms “isolated from, or substantially free of contaminatingmolecules” is understood to include a molecule containing less thanabout 20% contaminating molecules, based on dry weight calculations,preferably, less than about 5% contaminating molecules.

[0401] The terms “isolated” or “purified” do not refer to materials in anatural state, or materials separated into elements without furtherpurification. For example, separating a preparation of nucleic acids bygel electrophoresis, by itself, does not constitute purification unlessthe individual molecular species are subsequently isolated from the gelmatrix.

[0402] In one exemplary embodiment, a polynucleotide encoding apolypeptide of interest is ligated into a fusion polynucleotide encodinganother polypeptide which facilitates purification, for instance, apolypeptide with readily available antibodies, such as VP6 rotaviruscapsid protein, a vaccinia virus capsid protein, or the bacterial GSTprotein. When expressed, the facilitator polypeptide enablespurification of the polypeptide of interest and immunologicalidentification of host cells which express it. In the case of GST-fusionproteins, purification may be achieved by use of glutathione-conjugatedsepharose beads in affinity chromatographic techniques well known in theart (see, for instance, Ausubel 1998¹⁴⁰).

[0403] In a related exemplary embodiment, the fusion polypeptideincludes a polyamino acid tract, such as the polyhistidine/enterokinasecleavage site, which confers physical properties that inherently enablepurification. In this example, purification may be achieved throughnickel metal affinity chromatography. Once purified, the polyhistidinetract included to enable purification can be removed by treatment withenterokinase in vitro to release the polypeptide fragment of interest.

[0404] For molecules synthesized by an organism, for instance,polypeptides or polynucleotides synthesized by human subjects, in apreferred exemplary embodiment, a purified polynucleotide or polypeptideis free of other molecules synthesized by same organism, accomplished,for example, by expression of a human gene in a non-human host cell.

[0405] The following sections present standard protocols for theformulation of certain types of agents.

[0406] (2) Small Molecules

[0407] One aspect of the invention pertains to administration of a smallmolecule of interest, equivalent small molecules, or homologous smallmolecules, isolated from, or substantially free of contaminatingmolecules, as treatment of a chronic disease.

[0408] The following sections present standard protocols for formulationof small molecules.

[0409] (a) Production

[0410] Small molecules, organic or inorganic, may be synthesized invitro by any of a number of methods well known in the art. Those smallmolecules, and others synthesized in vivo, may by purified by, forinstance, liquid or thin layer chromatography, high performance liquidchromatography (HPLC), electrophoresis, or some other suitabletechnique.

[0411] (3) Polypeptides

[0412] Another aspect of the invention pertains to administration of apolypeptide of interest, equivalent polypeptides, or homologouspolypeptides, isolated from, or substantially free of contaminatingmolecules, as treatment of a chronic disease.

[0413] The following sections present standard protocols for theformulation of polypeptides.

[0414] (a) Production

[0415] (i) In vitro

[0416] In one exemplary embodiment, a polypeptide of interest isproduced in vitro by introducing into a host cell by any of a number ofmeans well known in the art (see protocols below) a recombinantexpression vector carrying a polynucleotide, preferably obtained fromvertebrates, especially mammals, encoding a polypeptide of interest,equivalents of such polypeptide, or homologous polypeptides. Therecombinant polypeptide is engineered to include a tag to facilitatepurification. Such tags include fragments of the GST protein, orpolyamino acid tracts either recognized by specific antibodies, or whichconvey physical properties facilitating purification (see also below).Following culture under suitable conditions, the cells are lysed and theexpressed polypeptide purified. Typical culture conditions includeappropriate host cells, growth medium, antibiotics, nutrients, and othermetabolic byproducts. The expressed polypeptide may be isolated fromeither a host cell lysate, culture medium, or both depending on theexpressed polypeptide. Purification may involve any of many techniqueswell known in the art, including but not limited to, gel filtration,affinity chromatography, gel electrophoresis, ion-exchangechromatography, and others.

[0417] Polynucleotides, both mRNA and DNA, can be extracted fromprokaryotic or eukaryotic cells, or whole animals, at any developmentalstage, for instance, adults, juveniles, or embryos. Polynucleotides maybe isolated, or cloned from a genomic library, cDNA library, or freshlyisolated nucleic acids, using protocols well known in the art. Forinstance, total RNA is isolated from cells, and mRNA converted to cDNAusing oligo dT primers and viral reverse transcriptase. Alternatively, apolynucleotide of interest may be amplified using PCR. In any case, theinitial nucleic acid preparation may include either RNA or DNA and theprotocols chosen accordingly. The resulting DNA is inserted into anappropriate vector, for instance, bacterial plasmid, recombinant virus,cosmid, or bacteriophage, using procedures well known in the art.

[0418] Nucleotide sequences are considered functionally linked if onesequence regulates expression of the other. To facilitate expression ofa polypeptide of interest, the cloning vector should include suitabletranscriptional regulatory sequences well known in the art, forinstance, promoter, enhancer, polyadenylation site, etc., functionallylinked to the polynucleotide expressing the polypeptide of interest. Inone exemplary embodiment, an expression vector is constructed to carry apolynucleotide, a naturally occurring sequence, a gene, a fusion of twoor more genes, or some other synthetic variant, under control of aregulatory sequence, such that when introduced into a cell expresses apolypeptide of interest.

[0419] Both viral and nonviral gene transfer methods may be used tointroduce desirable polynucleotides into cells. Viral methods exploitnatural mechanisms for viral attachment and entry into target cells.Nonviral methods take advantage of normal mammalian transmembranetransport mechanisms, for example, endocytosis. Exemplary protocolsemploy packaging of deliverable polynucleotides in liposomes, encasementin synthetic viral envelopes or poly-lysine, and precipitation withcalcium phosphate (see also below).

[0420] The variety of suitable expression vectors is vast and growing.For example, mammalian expression vectors typically include prokaryoticelements which facilitate propagation in the laboratory, eukaryoticelements which promote and regulate expression in mammalian cells, andgenes encoding selectable markers. The list of appropriate vectorsincludes, but is not limited to, pcDNA/neo, pcDNA/amp, pRSVneo, pZIPneo,and a host of others.

[0421] Many viral derivatives are also available, for instance, pHEBo,derived from the Epstein-Barr virus, BPV-a derived from the bovinepapillomavirus, and the pLRCX system (BD Biosciences Clonetech, Inc.).The use of mammalian expression vectors is well known in the art (see,for example, Sambrook 2001, ibid, chapters 15 and 16). Similarly, manyvectors are available for expression of recombinant polypeptides inyeast, including, but not limited to, YEP24, YEP5, YEP51, pYES2. The useof expression vectors in yeast is well known in the art.

[0422] In addition to mammalian and yeast expression systems, a systemof vectors is available which permits expression in insect cells. Thesystem, derived from baculoviruses, includes pAcUW-based vectors (forinstance, pAcUW1), pVL-based vectors (for instance, pVL1292 andpVL1393), and pBlueBac-based vectors which carry the gene encodingβ-galactosidase to facilitate selection of host cells harboringrecombinant vectors.

[0423] (ii) In situ

[0424] In another exemplary embodiment, a polypeptide of interest isexpressed in situ by administering to an animal or human subject by anyof a number of means well known in the art (see protocols below) arecombinant expression vector carrying a polynucleotide encoding thepolypeptide of interest, equivalent polypeptides, or homologouspolypeptides.

[0425] In the present invention, such vectors may be used as therapeuticagents to introduce polynucleotides into cells that express constructiveor disruptive polypeptides (for exemplary applications see, forinstance, Friedmann 1999¹⁴¹).

[0426] It is critical that the potential effects of microcompetitionbetween the enhancer, or other polynucleotide sequences carried in thedelivery vector, and cellular genes be considered and manipulated whereneeded. As an example consider a case where the polypeptide of interestbinds an enhancer carried by the vector, for instance, a delivery vectorthat expresses GABP under control of a promoter that includes an N-box.In one exemplary embodiment, the vector expresses, in situ, a highenough concentration of the polypeptide of interest such that anybinding of the polypeptide to the enhancer sequences within the vectoritself is negligible. In other words, the vector expresses enough freepolypeptides to produce the desired biological activity in treatedcells. In another example, the polypeptide is not a transcriptionfactor, but the delivery vector carries a polynucleotide thatmicrocompetes with cellular genes for a cellular transcription factor,for instance, a vector that expresses Rb and microcompetes with cellulargenes for GABP. In an exemplary embodiment, the delivery vector alsoincludes a polynucleotide sequence that expresses the microcompetedtranscription factor, or is delivered in conjunction with another vectorthat expresses the microcompeted transcription factor. In the example,the Rb vector includes a sequence that expresses GABP, or is deliveredin conjunction with a vector that expresses GABP.

[0427] (4) Polynucleotides

[0428] Another aspect of the invention pertains to administration of apolynucleotide as antisense/antigene, ribozyme, triple helix, homologousnucleic acids, peptide nucleic acids, or microcompetitiors, equivalentpolynucleotides, or homologous polynucleotides, isolated from, orsubstantially free of contaminating molecules, as treatment for achronic disease.

[0429] The following sections present standard protocols for theformulation of such polynucleotides. Since antisense/antigene, ribozyme,triple helix, homologous nucleic acids, peptide nucleic acids, andmicrocompetition agents are nucleic acid based, they share protocols fortheir synthesis, mechanisms of delivery and potential pitfalls in theiruse including, but not limited to, susceptibility to extracellular andintracellular nucleases, instability and the potential for nonspecificinteractions. In consideration of these common issues, the generalmethods for the formulation and delivery, as well as caveats regardingthe use of nucleic agents, described first, apply similarly to eachsubsequent agent.

[0430] (a) Antisense/Antigene

[0431] In the present invention, the terms “antisense” and “antigene”polynucleotides is understood to include naturally or artificiallygenerated polynucleotides capable of in situ binding to RNA or DNA,respectively. Antisense binding to mRNA may modify translation of boundmRNA, while antigene binding to DNA may modify transcription of boundDNA. Antisense/antigene binding may modify binding of a polypeptide ofinterest to RNA or DNA, for instance binding of an antigene to a foreignN-box may reduce binding of cellular GABP to the foreign N-box resultingin attenuated microcompetition between the foreign polynucleotide and acellular gene for GABP. Antisense/antigene binding may also modify,i.e., decrease or increase, expression of a polypeptide of interest.

[0432] Binding, or hybridization of the antisense/antigene agent, may beachieved by base complementarity, or by interaction with the majorgroove of the cellular DNA duplex. The techniques and conditions forachieving such interactions are well known in the art.

[0433] The target of antisense/antigene agents has been thoroughlystudied and is well known in the art. For instance, the antisensepreferred target is the translational initiation site of a gene ofinterest, from approximately 10 nucleotides upstream to approximately 10nucleotides downstream of the translational initiation site.Oligonucleotides targeting the 3′ untranslated mRNA regions are alsoeffective inhibitors of translation. Therefore, oligonucleotidestargeting the 5′ or 3′ UTRs of a polynucleotide of interest may be usedas antisense agents to inhibit translation. Antisense agents targetingthe coding region are less effective inhibitors of translation but maybe used when appropriate.

[0434] Effective synthetic agents are typically between 20 and 30nucleotides in length. However, to be effective, a complementarysequence must be sufficiently complementary to bind tightly and uniquelyto the polynucleotide of interest. The degree of complementarity isgenerally understood by those skilled in the art to be measured relativeto the length of the antisense/antigene agent. In other words, threebases of mismatch in a 20 base oligonucleotide has a more profoundlydetrimental effect than three bases of mismatch in a 100 baseoligonucleotide. Inadequate complementarity results in ineffectiveinhibition, or unwanted binding to sequences other than thepolynucleotide of interest. In the latter case, inadvertent effects mayinclude unwanted inhibition of genes other than a gene of interest.Specificity and binding avidity are easily determined empirically bymethods known in the art.

[0435] Several methods are suitable for the delivery ofantisense/antigene agents. In one exemplary embodiment, a recombinantexpression plasmid is engineered to express antisense RNA followingintroduction into host cells. The RNA is complementary to a uniqueportion of DNA or mRNA sequence of interest. In an alternativeembodiment, chemically derivatized synthetic oligonucleotides are usedas antisense/antigene agents. Such oligonucleotides may contain modifiednucleotides to attain increased stability once exposed to cellularnucleases. Examples of modified nucleotides include, but are not limitedto, nucleotides carrying phosphoramidate, phosphorothioate andmethylphosphonate groups.

[0436] Whichever sequence of the polynucleotide of interest is targetedby antisense/antigene agents, in vitro studies should be undertakenfirst to determine the effectiveness and specificity of the agent.Control treatments should be included to differentiate between effectsspecifically elicited by the agent and non-specific biological effectsof the treatment. Control polynucleotides should have same length andnucleotide composition as the agent with the base sequence randomized.

[0437] Antisense/antigene agents can be oligonucleotides of RNA, DNA,mixtures of both, chemical derivatives of either, and single or doublestranded. Nucleotides within the oligonucleotide may carry modificationson the nucleotide base, the sugar or the phosphate backbone. Forexample, modifications to the nucleotide base involves a number ofcompounds including, but not limited to, hypoxanthine, xanthine,2-methyladenine, 2-methylguanine, 7-methylguanine, 5-fluorouracil,3-methylcytosine, 2-thiocytosine, 2-thiouracil, 5-methylcytosine,5-methylaminomethyluracil, and a host of others well known in the art.Modifications are generally incorporated to increase stability, e.g.infer resistance to cellular nucleases, stabilize hybridization, orincrease solubility of the agent, increased cellular uptake, or someother appropriate action.

[0438] In a related exemplary embodiment, adducts of polypeptides, totarget the agent to cellular receptors in vivo, or other compounds whichfacilitate transport into the target cell are included. Additionalcompounds may be adducted to the antisense/antigene agent to enablecrossing of the blood-brain barrier, cleavage of the target sequenceupon binding, or to intercalate in the duplex which results fromhybridization to stabilize that complex. Any such modification, intendedto increase effectiveness of the antisense/antigene agent, is includedin the present invention.

[0439] Similarly, the antisense/antigene agent may include modificationsto the phosphate backbone including, but not limited to,phosphorothioates, phosphordamidate, methylphosphonate, and others. Theagent may also contain modified sugars including, but not limitedvariants of arabinose, xylulose and hexose.

[0440] In another exemplary embodiment, the antisense/antigene agent isan alpha anomeric oligonucleotide capable of forming parallel, ratherthan antiparallel, hybrids with a cellular mRNA of interest.

[0441] It is common for antisense agents to be targeted against thecoding regions of an RNA of interest to effect translational inhibition.In a preferred embodiment, antisense agents are targeted instead againstthe transcribed but untranslated region of an RNA transcript. In thiscase, rather than achieving translational inhibition, it is likely thatoligonucleotides hybridized to the target transcript will lead to mRNAdegradation through a pathway mediated by RNaseH or similar cellularenzymes.

[0442] For optimal efficacy, the antisense/antigene agents must bedelivered to cells carrying the polynucleotide of interest in vivo.Several delivery methods are known in the art, including but not limitedto, targeting techniques employing polypeptides linked to theantisense/antigene agent which bind to specific cellular receptors. Inthis instance the agents may be provided systemically. Alternatively theagents may be injected directly into the tissue of interest, or packagedin a virus, including retroviruses, chosen because its host rangeincludes the target cell. In every case, the agent must enter the targetcell to be effective.

[0443] Antisense/antigene methodologies often face the problem ofachieving sufficient intracellular concentration of the agent toeffectively compete with cellular transcription and/or translationfactors. To overcome this challenge, those skilled in the art introducerecombinant expression vectors carrying the antisense/antigene agent.Once introduced into the target cell, expression of theantisense/antigene agent from the incorporated RNA polymerase II or IIIpromoter results in sufficient intracellular concentrations. Vectors canbe chosen to integrate into the host cell chromosomes, thereby becomingstable through multiple rounds of cell division, or vectors may be usedwhich remain un integrated and therefore are lost when the target celldivides. In either case, the primary goal is attaining levels oftranscription that produce sufficient antisense/antigene agents to beeffective. The choice of a suitable vector and the development of aneffective antisense construct involves techniques standard in the art.

[0444] Antisense/antigene expression man be regulated by any promoterknown to be active in mammalian, especially human, cells and may beeither constitutively active or inducible. Regardless of the promoterchosen, it is important to test for the effect of any enhancer regionsintrinsic to those promoters as they may participate in microcompetitionwith cellular genes. In the case of inducible promoters, the biologicaleffects of the expressed antisense can be discerned from any effect thepromoter has on microcompetition by assaying any bioactivity with andwithout induced gene expression. Suitable promoters, inducible or not,are well known in the art (see, for example, Jones 1998¹⁴²).

[0445] Antisense agents may be prepared using any of a number of methodscommonly known to those skilled in the art. In on exemplary embodiment,oligonucleotides, up to approximately 50 nucleotides in length, may besynthesized using automated processes employing solid phase, e.g.controlled pore glass (CPG) technology, such as that used on the AppliedBiosystems model 394 medium throughput synthesizer, or 5′-phosphate ON(cyanoethyl phosphoramidite) chemistry developed by ClonotechLaboratories, Inc. In each of these procedures, oligonucleotides aresynthesized from a single nucleotide using a series of deprotection andligation steps. The underlying chemistry of the reactions is standardpractice and the availability and accessibility of automatedsynthesizers bring these synthetic technologies within the grasp ofanyone skilled in the art.

[0446] Despite the ease of synthesis, the selection of effectiveantisense agents involves the identification of a suitable target forthe agent. This process is simplified somewhat by the many softwareprograms available, such as, for example, Premier Primer 5, availablefrom Premier Biosoft International or Primer 3, available online athttp://www-genome.wi.mit.edu/cgi-bin/primer/primer3.cgi. Alternatively,antisense agents may be designed manually by a scientist skilled in theart. Relevant aspects of the design process which need attention includeselection of the target region to which the antisense agent will bind.Ideally it will be the gene promoter, if the target is DNA, or thetranslation initiation site if the target is an mRNA. Attention alsoneeds to be paid to the length of the agent, typically at least 20nucleotides are needed for specificity. Shorter oligonucleotides carrythe risk of non-specific binding and therefore may lead to undesiredside effects. Also, the agents must be composed of a sequence that willnot promote hybridization between the oligonucleotides in the agentduring application. Taken together, these considerations are well knownand are addressed by standard procedures well known in the art.

[0447] Longer antisense agents may be produced within the target cellfrom recombinant expression vectors. In one exemplary embodiment, thedesired antisense-encoding sequences can be incorporated into anappropriate expression vector selected because it contains theregulatory sequences necessary to ensure expression in the target celltype. Selection of the sequence composition of the antisense agent musttake into account the same considerations used to design shorteroligonucleotides as described in the previous paragraph including, butnot limited to, binding specificity for the target sequence andminimizing interactions between the expressed agents. Techniques for thedesign and construction of appropriate recombinant expression vectorsare well known to those skilled in the art.

[0448] Control agents, whether synthetic oligonucleotides or longerantisense agents expressed in vivo by expression vectors, are employedto validate the efficacy and specificity of the therapeutic agents. Eachcontrol agent should have the same nucleotide composition and length asthe therapeutic agent but the sequence should be random. Employment ofthis agent will permit the determination of whether any effects observedafter treatment with the therapeutic agent are indeed specific.Specificity will reduce the potential for binding to targets other thanthose desired, thereby reducing associated unwanted side effects.

[0449] Purification of Oligonucleotides: The efficacy of syntheticoligonucleotide agents is impacted by their purity. Under typicalconditions, approximately 75% of the synthesis products are full lengthwhile the remaining 25% of the oligonucleotides are shorter. Thisproportion of full length to shorter products varies with the length ofthe desired product. The synthesis of longer oligonucleotides is lessefficient, and therefore the synthesis products contain a smallerproportion of full length products, than that of shorter ones. Unwanted,shorter synthesis products have reduced specificity compared to the fulllength products and are therefore undesirable in a therapeuticformulation due to their reduced specificity which in turn leads to anincreased risk of side effects.

[0450] In one exemplary embodiment, full length oligonucleotides greaterthan 50 bp in length are purified by virtue of their size. Gelpermeation chromatography is used to separate full length products fromthe shorter synthetic byproducts. In a complementary exemplaryembodiment, full length synthetic oligonucleotides shorter than 50 bpmay be purified by liquid chromatography using charged resins such ashydroxyapatite or nucleic acid specific resins such as RPC-5 (which iscomposed of trioctylmethylamine adsorbed onto hydrophobic plasticparticles). This latter technique exploits both hydrophobic and ionexchange methods to achieve high reagent purity and is amenable to usein HPLC.

[0451] Regardless of the method of purification used, the desiredoligonucleotides are concentrated by precipitation with ice cold ethanolfollowed by lyophilization and dissolution in an appropriate carrier fortreatment. Carrier selection is another important component of agentformulation. It is essential that the carrier used is first tested forbiological activity in the target cell type. This control measure, wellknown to those skilled in the art, will ensure that any effects observedupon administration of the nucleic acid agent are indeed due to theagent and not the carrier in which it is administered (on purificationof oligonucleotides see, for instance, Deshmukh (1999¹⁴³).

[0452] Delivery of Oligonucleotides: Methods for effectiveadministration of antisense agents vary with the agent used. In oneexemplary embodiment, synthetic oligonucleotides are delivered by simplediffusion into the target cells. Advantages of this delivery methodinclude the ability to administer the agent systemically, for example byintravenous injection. This method, while effective carries severalrisks, not the least of which is the potential to introduceoligonucleotides into cells other than those of the desired target.Another disadvantage involves the risk of degradation by nucleases inblood and interstitial fluid. This second disadvantage may be partiallyavoided by modification of the synthetic oligonucleotide in such a way,for example by incorporated modified nucleotides such as those carryingphosphorothioate or methyl phosphonate moieties, which renders themrelatively resistant to exonuclease degradation.

[0453] In a related embodiment those same agents may be delivered by wayof liposome mediated transfection as described by Daftary and Taylor(2001¹⁴⁴) This method enhances diffusion into the target cell byencasing the antisense agent in a lipophilic liposome. However, thismethod too has drawbacks. While cellular uptake is enhanced, the ratioof liposome components to DNA must be carefully controlled in order tomaximize delivery efficiency. This technique is commonly employed and iswell known to those skilled in the art.

[0454] In another exemplary embodiment, antisense expressing viralvectors may be used to confer target cell specificity. In some cases,viral delivery agents may be selected which include the target cell typein their respective host range. This delivery method minimizes unwantedside effects that otherwise may arise from delivery of the therapeuticagent to the incorrect cell type. However, this advantage may be negatedif the multiplicity of infection is too high and non-specific infectionis thereby promoted. This potential problem may be avoided by thoroughlytesting any viral deliver agent, using techniques well known in the art,prior to its clinical administration.

[0455] (b) Ribozymes

[0456] While antisense agents act by either inhibiting transcription ortranslation of the target gene, or by inducing enzyme-mediatedtranscript degradation by RNase H or a similar enzyme, ribozymes offeran alternative approach. Ribozymes are RNA molecules which natively bindto and cleave target transcripts. Typical ribozymes bind to and cleaveRNA at specific sites, however hammerhead ribozymes cleave targettranscripts at sites directed by flanking nucleotide sequences whichbind to the target site. The use of hammerhead ribozymes is preferredbecause the only sequence requirement for their activity is the UGdinucleotide arranged in the 5′-3′ orientation. Hammerhead technologiesare well known in the art (see, for example Doherty 2001¹⁴⁵, orGoodchild 2000¹⁴⁶). In a preferred embodiment, the sequence targeted bythe ribozyme lies near the 5′ end of the transcript. That will resultcleavage of the transcript near the translation initiation site therebyblocking translation of a full-length protein.

[0457] Ribozymes identified in Tetrahymena thermophila, which employ aneight base pair active site which duplexes with the target RNA molecule,are included in this invention. This invention includes those ribozymes,described and characterized by Cech and coworkers (i.e. IVS or L-19IVSRNA), which target eight base-pair sequences in a gene of interest andany others which may be effective in inhibiting expression of adisrupted gene or a gene in a disrupting pathway. For the catalyticsequence of these agents see, for instance, U.S. Pat. No. 5,093,246,incorporated entirely herein by reference. Any ribozyme or hammerheadribozyme molecules that targets RNA sequences expressed by a foreignpolynucleotide, disrupted gene or gene in a disrupted pathway areincluded in this invention.

[0458] Ribozymes, being RNA molecules of specific sequence, may besynthesized with modified nucleotides which enable better targeting tothe host cell of interest or which improve stability. As described abovefor conventional antisense agents, the preferred method of deliveryinvolves introduction into the target cell, a recombinant expressionvector encoding the ribosome. Inclusion of an appropriatetranscriptional promoter will ensure sufficient expression to cleave anddisrupt transcripts of foreign DNA or disrupted genes or genes in adisrupting pathway. The catalytic nature of ribozymes permits theireffective use at concentrations below those needed for traditionalantisense agents.

[0459] Identification of ribozyme cleavage sites within a transcript ofinterest is accomplished with any of a number of computer algorithmswhich scan linear oligonucleotide sequences for alignments with a querysequence. The identified sequence, commonly containing the trinucleotidesequences GUC, GUA or GUU, will serve as the nucleus of a longersequence of approximately 20 nucleotides in length. That longer sequencewill be examined, again with appropriate computer algorithms well knownin the art, for their potential to form secondary structures which mayinterfere with the action of targeted ribozyme agents. Alternatively,empirical assays employing ribonucleases may be used to probe theaccessibility of identified target sequences.

[0460] Ribozymes comprise a unique class of oligonucleotides which bindto specific ribonucleic acid targets and promote their hydrolysis. Thedesign of ribozyme agents is well known to those skilled in the art. Inorder to prepare effective ribozyme agents, initially a suitable targetsequence must be identified which confers specificity to the agent inorder to minimize unwanted side effects and maximize efficacy. Once thattarget is identified the ribozyme agent is synthesized using standardoligonucleotide synthesis procedures such as those exemplified herein.Delivery to the target cell may be accomplished by direct transfectionex vivo or by liposome-mediated transfection.

[0461] Ensuring the purity and efficacy of ribozyme agents may be moreimportant than for other nucleic acid agents because their intendedeffects, namely the hydrolysis of target sequences, are irreversible. Inthis light extensive preclinical testing is essential to minimizeunwanted side effects. These risks are, however, outweighed by thepotential effectiveness of ribozyme agents.

[0462] (c) Triple Helix

[0463] In a related embodiment, synthetic single-strandeddeoxyribonucleotides can be chosen which form triple helices accordingto the Hoogsteen base pairing rules. The rules necessitate longstretches of either purines or pyrimidines on one strand of the DNAduplex. In either case, triplexes are formed, with pyrimidines pairingwith purines within the target sequence and vice versa, which inhibittranscription of the target sequence. The effectiveness of a targetedtriplex forming oligonucleotide may be enhanced by including a“switchback” motif composed of alternating 5′-3′ and 3′-5′ regions ofpurines and pyrimidines. This “switchback” reduces the length of therequired purine or pyrimidine tract in the target because theoligonucleotide can form duplexes alternatively with each strand of thetarget sequence.

[0464] Triple helix forming agents are oligonucleotides which have beendesigned to interact with cellular nucleic acids and form triplehelices. The resulting structure may be targeted by intracellulardegradation pathways or may provide a steric block to nucleic acidreplication, transcription or translation depending on the target.

[0465] Triplex agent formulation begins with selection of an appropriatetarget sequence within the cells to be treated. That target may bewithin the cellular DNA or RNA or within that of an exogenous sourcesuch as an infecting virus. Suitable target sequences should containlong stretches of homopyrimidines or homopurines and the most effectivetargets contain alternative stretches of each. If the target is doublestranded DNA, the most effective targets surround and include thetranscriptional regulatory regions. Formation of a triplex between theagent and the target will inhibit the binding of RNA polymerase or otherrequisite transcriptional regulatory factors which otherwise bind thepromoter and upstream regulatory regions.

[0466] Triplex agents may be synthesized to be more resistant tocellular and extracellular nucleases by the inclusion of modifiednucleotides such as those containing phosphorothioate or methylphosphonate groups. In the event that such modifications interfere withbase pairing, additional adducts, such as derivatives of the baseintercalating agent acridine, may be incorporated into the therapeuticagent to restore desirable binding properties to the triplex formingoligonucleotide. Alternatively, if the intracellular target is an mRNA,C-5 propyne pyrimidines may be included in the syntheticoligophosphorothioate agent to increase its binding affinity for mRNAand therefore decrease the concentration required for effectiveness.

[0467] The affinity of triplex agents for their respective targets maybe assessed by electrophoretic gel retardation assays. The formation oftriplex structures will retard migration through an electrophoretic gel.Similarly, the stability of any triplex agent binding to its target canbe assessed by UV melting experiments. In these assays triplex agentsare mixed with their intended target in vitro and the resultingtriplexes are heated (with, for example, a Haake cryothermostat) whilemonitoring their UV absorbance (with, for example, a Kontron-Uvikon 940spectrophotometer) (on design of triplex forming oligonucleotides see,for instance, Francois (1999¹⁴⁷)).

[0468] Triplex forming agents are simply oligonucleotides designed toform triple helices with the target intracellular nucleic acid.Accordingly, their synthesis, purification and delivery parallels theprocedures described herein for other oligonucleotide agents. Each ofthese processes is commonly known to those skilled in the art.

[0469] (d) Homologous Recombination Agents

[0470] Binding of factors to foreign polynucleotides (either DNA orRNA), or polynucleotides of disrupted genes, or polynucleotides of agene in a disrupted or disrupting pathway, or expression of a foreigngene, or a disrupted gene, or a gene in a disrupted or disruptingpathway can also be reduced by mutating the DNA, inactivating, or“knocking out” the gene or its promoter using targeted homologousrecombination.

[0471] In one exemplary embodiment, a polynucleotide of interest flankedby DNA homologous to the polynucleotide interest (encompassing eitherthe coding or regulatory regions of the polynucleotide) can beintroduced into cells carrying the same sequence. Homologousrecombination mediated by the flanking sequences disrupts expression ofthe polynucleotide of interest and result in reduced expression. Thetechnique is frequently used by those skilled in the art to engineertransgenic animals that produce offspring with same disruption. However,the same approach may be used in humans by administering the engineeredconstruct into target cells. Regardless of expression vector platformchosen, it is important to recognize and control for anymicrocompetition effects that may be elicited by transcriptionalenhancers carried by the viral vectors (see also above). Controlexperiments must be carried out which study the biological activity of anon-recombinant viral vector to reveal any effects its intrinsicenhancers have on the target biological activities.

[0472] Nucleic acid agents for homologous recombination are designed tointeract with specific cellular DNA targets and undergo recombination.The specificity of the therapeutic agent is conferred by the nucleotidesequences at its termini, they must be complementary to adjacentcellular targets and bind them through Watson-Crick base pairing.

[0473] Formulation of these agents involves careful selection of thedesired cellular target. The nucleotide sequence of that target must beavailable in public or private sequence databases. The agent itself maybe comprised of a synthetic oligonucleotide or a recombinant nucleicacid carried in a suitable vector.

[0474] In one exemplary embodiment, a synthetic oligonucleotide may beused for homologous recombination in order to interrupt the codingsequence or regulatory sequences of the target gene. The oligonucleotideis designed to include nucleotides at its termini which arecomplementary to those of the target sequence and the central regionsmay contain any sequence that is neither complementary to the targetsequence nor carry an in-frame insertion into the target sequence.

[0475] In a related embodiment, a longer sequence of nucleic acid may beused. The sequence of interest, which is intended to either interrupt acellular gene or insert additional coding capacity into it, is flankedby sequences homologous to the cellular target. That entire DNA fragmentis then inserted into an appropriate prokaryotic or viral vector fordelivery to the target cells. Once inside the cell the agent will bindto and recombine with the target gene.

[0476] (e) Peptide Nucleic Acids

[0477] In various embodiments, hybridization of the nucleic acid agentsdescribed herein may be enhanced by the substitution of amino acids forthe deoxyribose of the nucleic acid backbone. This substitution, therebycreating peptide nucleic acids (see, for example, Hyrup 1996¹⁴⁸). Thismodification leads to a reduction of the overall negative charge on thebackbone and therefore reduces the need for counter ions to permitsequence-specific hybridization of two strands of negatively chargedpolynucleotides. Peptide nucleic acids can be synthesized usingtechniques well known in the art such as the solid phase protocolsdescribed by Hyrup and Nielsen (1996, ibid), and Perry-O'Keefe 1996¹⁴⁹,included herein in their entirety by reference.

[0478] Oligonucleotides so modified can be used in the same therapeutictechniques as unmodified homologs. They can be used as antisense agentsdesigned to interfere with the expression of a foreign polynucleotide, adisrupted gene, or a gene in a disrupted pathway. Similarly, by virtueof their enhanced hybridization qualities, peptide nucleic acids can beused, for example, as primers for the PCR, for S1 nuclease mapping ofsingle stranded regions and for other enzyme-based techniques.Similarly, peptide nucleic acids may be modified by the addition oflipophilic moieties to enhance the cellular uptake of therapeuticoligonucleotide agents. In related embodiments, peptide nucleotideagents may be synthesized as chimeras comprised of peptide nucleic acidsand unmodified DNA. This configuration exploits the advantages of apeptide nucleic acid while the DNA portion of the molecule can serve asa substrate for cellular enzymes.

[0479] Peptide Nucleic Acid (PNA) is a DNA analog in which thesugar-phosphate backbone contains a pseudopeptide rather than the sugarscharacteristic of DNA. Like DNA, PNA agents bind complementary nucleicacid strands thereby mimicking the behavior of DNA. This activity isenhanced by the neutral, rather than negatively charged, backbone of PNAwhich promotes more tenacious and more specific binding than that ofDNA. These are among many favorable properties of PNA and include, inaddition, increased stability and exhibit improved hybridizationproperties compared to their DNA analogs. While the mechanism of PNAaction is currently not fully understood, for example PNA-RNA hybridsare not targets for RNase H degradation as are DNA-RNA hybrids, it islikely that they inhibit translation by blocking the binding of RNApolymerase or other critical factors to the target mRNA.

[0480] In this light, it is important to select targets which includethe translation initiation codon. Other target sites further downstreamon the mRNA may be effective at inhibiting translation by interferingwith ribosome transit although the role of this activity will need to bedetermined empirically for each agent developed. In any case the actualmechanism of action, while interesting, is not necessary to ascertain aslong as the agent is effective and does not induce undesired sideeffects.

[0481] Homopurines are best targeted by homopyrimidine PNAs withstretches of greater than 8 bp providing suitable targets within doublestranded DNA. The synthesis of PNA agents is achieved using automatedsolid-phase techniques employing Boc-, Fmoc- or Mmt-protected monomers.Alternatively, commercial sources of custom synthetic PNAs, includingApplied Biosystems (Foster City, Calif.) may be exploited to minimizein-house expenses and expertise (on design of PNA see, for instance,Nielsen 1999¹⁵⁰)

[0482] (5) Antibodies and Antigens

[0483] Another aspect of the invention pertains to the administration ofan antibody of interest, equivalent of such antibody, homolog of suchantibody, as treatment of a chronic disease.

[0484] For example, using standard protocols, one skilled in the art canuse immunogens derived from a foreign polynucleotide, foreignpolypeptide, disrupted gene, disrupted polypeptide, gene or polypeptidein a disruptive or disrupted pathway, to produce anti-protein,anti-peptide antisera, or monoclonal antibodies (see, for example,Harlow and Lane 1999¹⁵¹, Sambrook 1989¹⁵²).

[0485] Animals which have been injected with an immunogenic agent canserve as sources of antisera containing polyclonal antibodies.Monoclonal antibodies, if desired, may be prepared by isolatinglymphocytes from the immunized animals and fusing them, in vitro withimmortal, oncogenically transformed cells. Clonal lines from theresulting somatic cell hybrids, or hybridomas, can be used as sources ofmonoclonal antibodies specific for the immunogen of interest. Techniquesfor developing hybridomas and for isolating and characterizingmonoclonal antibodies are well known in the art (see for instance,Kohler 1975¹⁵³ and Zola 2000¹⁵⁴).

[0486] In the context of this invention, “antibody” refers to entiremolecules or their fragments which react specifically with polypeptidesor polynucleotides of interest, whether they are monospecific,bispecific or chimeras which recognize more than two antigenicdeterminants. Those skilled in the art employ well known methods forproducing specific antibodies and for fragmenting same. While severalmethods are known to produce antibody fragments, pepsin, for example, isused to treat whole antibody molecules to produce F(ab)₂ fragments.These fragments can be further dissociated with chemicals, such as betamercaptoethanol or dithiothreotol, which reduce intra and intermoleculardisulfide bridges resulting in the release of Fab fragments.

[0487] Once produced, isolated and characterized, antibodies, orfragments thereof, which bind to antigenic determinants of interest maybe used for diagnostic and analytical purposes. For example, they may beused in immunohistochemical assays to assess expression levels ofpolynucleotides or polypeptides of interest. They may also be employedin other immunoassays, including but not limited to, Western blots,immunoaffinity chromatography, and immunoprecipitation carried out toquantify protein levels in cells or tissues of interest. The assays,individually or together, may also be used by one skilled in the art tomeasure the concentration a protein of interest before and after therapyto assess therapeutic efficacy.

[0488] Similarly, it is common in the art to use specific antibodies toscreen libraries of recombinant expression vectors for those expressinga protein or polypeptide of interest. Suitable expression vectors arecommonly derived from bacteriophage, including, for example, λgt11 andits derivatives. Identification of expression vectors, from among alibrary of similar recombinants, can lead to the identification ofvectors expressing a polypeptide of interest which may then itself beused in diagnostic or therapeutic assays. In a preferred embodiment,antibodies specific for a particular polypeptide, protein or antigenicdeterminant carried thereon, will cross react with homologouscounterparts from different species to facilitate antibodycharacterization and assay development.

[0489] Antibodies may serve as effective therapeutic agents for theinactivation of specific cellular proteins or for targeting othertherapeutic agents to cells expressing particular surface antigens towhich an antibody may bind. Polyclonal antibodies are prepared in asuitable host organism, typically rabbit, goat or horse, by injectingthe appropriate purified antigen into the host. Following a regimen ofrepeated challenges by the desired antigen, using protocols well knownto those skilled in the art, serum is drawn from the host and assayedfor the presence of antibodies. Once a suitable response is detected,additional serum is removed, perhaps leading to exsanguination of theproducing organism, and the desired antibodies are purified.

[0490] Monoclonal antibodies may be prepared by any number of techniqueswell known to those skilled in the art. In one exemplary embodiment,cells expressing the desired target antigen are fused with immortalizedcells in vitro. The resulting hybridomas are cultured and clonal linesare derived using standard tissue culture techniques. Each resultingclone is assayed for expression of antibodies against the desiredantigen, typically but not necessarily by ELISA.

[0491] Antibodies may be purified by a number of chromatographictechniques. In one exemplary embodiment, antibodies may be bound to S.aureus protein A cross-linked to a suitable support resin (e.g.sepharose). The crude antibody preparation is slowly applied to thechromatographic column under conditions which permit antibody-protein Ainteractions. The resin is then washed with several column volumes ofbuffer to remove adventitiously bound and trapped proteins, leaving onlyspecifically bound antibodies on the column. Those are eluted by washingthe column with 100 mM glycine (pH 3.0) and monitoring protein elutionspectrophotometrically.

[0492] In an alternative embodiment, antibodies are purified by bindingto an affinity column comprised of antigen cross-linked to anappropriate solid support. Bound antibodies may be eluted by any of anumber of methods and may include the use of an elution buffercontaining glycine at low (e.g. 3.0) pH or 3M potassium thiocyanate and0.5M NH₄OH. Due to the varied mechanisms involved with antibody-antigeninteractions, the actual optimal elution conditions must determinedempirically.

[0493] The therapeutic efficacy of polyclonal compared to monoclonalantibodies cannot be predicted. Each has strengths and weaknesses. Forexample, polyclonal antibodies necessarily target multiple antigenicdeterminants on the target antigen. This feature may increase reactivitybut, at the same time, may decrease specificity. On the other hand,monoclonal antibodies are exquisitely specific for a single antigenicdeterminant on the target antigen. This specificity greatly reduces therisk of unwanted reactivity with other antigens, and the associated sideeffects, yet carries the risk that the target antigenic determinant maybe inaccessible in the cellular environment, either due to the naturalfolding of the protein or through interactions with other cellularmolecules. In every case, the efficacy of any antibody agent must bedetermined empirically using a variety of techniques well known to thoseskilled in the art.

[0494] Antibody production is necessarily preceded by the isolation andpurification of appropriate antigens. Cellular proteins may be purifiedby any of a number of techniques well known to those skilled in the art.In one exemplary embodiment, cells expressing the desired antigen arelysed in the presence of non-ionic detergents and the resulting lysateis subjected to purification. That lysate is then fractionated byprecipitation in the presence of ammonium sulfate. Sequentially higherconcentrations of ammonium sulfate are used to derive protein mixtureswhich differ by their solubility in ammonium sulfate. Each fraction isthen assessed for the presence of the desired antigen.

[0495] The fraction carrying the protein of interest is subjected tofurther purification by any of a number of well known methods. Forinstance, if an antibody against the protein is available, the proteinmay be purified by affinity chromatography using a resin of substrate,typically sepharose, dextran or some similar insoluble polymer, to whichthe antibody is conjugated. The protein mixture containing the desiredantigen is exposed to the resin under conditions which promoteantibody-antigen interactions. Adventitiously bound proteins are washedfrom the resin with an excess of binding buffer and the antigens areeluted with buffer containing an ionic detergent such as sodiumdodecylsulfate (SDS).

[0496] In an alternative embodiment, crude fractions of cellularproteins are further purified using methods well known in the artinvolving ion exchange or molecular exclusion chromatographictechniques. The purity of antigens isolated by any technique may beassessed by electrophoresis through denaturing polyacrylamide gelsfollowed by visualization by staining.

c) Assay Protocols

[0497] One aspect of the invention pertains to assaying the effect of anagent on a molecule of interest, equivalent molecules, or homologousmolecules during drug discovery, development, use as treatment, orduring diagnosis.

[0498] (1) Definitions

[0499] (a) Molecule of Interest

[0500] The term “molecule of interest” is understood to include, but notlimited to, p300/cbp, p300/cbp polynucleotides, p300/cbp factors,p300/cbp regulated genes, p300/cbp regulated polypeptides, p300/cbpfactor kinases, p300/cbp factor phosphatases, p300/cbp agents, foreignp300/cbp polynucleotides, p300/cbp viruses, disrupted genes, disruptedpolypeptides, genes in disrupted pathways, polypeptides in disruptedpathways, genes in disruptive pathways, polypeptides in disruptivepathways.

[0501] Every gene and protein mentioned in this invention is uniquelydefined by its sequence as published in public databases. See, forinstance, the sequences in the nucleotide and protein sequence databasesat NCBI (also known as Entrez, the name of the search and retrievalsystem), GenBank, the NIH genetic sequence database, DDBJ, the DNADataBank of Japan, EMBL, the European Molecular Biology Laboratorydatabase (GenBank, DDBJ and EMBL comprise the International NucleotideSequence Database Collaboration), SWISS-PROT, the protein knowledgebase,and TrEMBL, the computer-annotated supplement to SWISS-PROT (see alsothe search and retrieval system Expasy), PROSITE, the database ofprotein families and domains, and TRANSFAC, the database oftranscription factors. By a gene it is meant the coding and non codingregions, the promoters, enhancers, and the 5′ and 3′ UTRs. Publishedsequences are considered standard information and are well known in theart. In one exemplary embodiment, sequences for certain genes andproteins of interest in this invention are listed in the followingsection. For most genes, the list includes the human sequence. However,homologous sequences (see definition below) are available in the abovedatabases for other organisms, such as mouse, rat, etc. The followinglisted sequences should be regarded as illustrations, and, therefore,should not be construed as limiting the invention in any way.

[0502] List of Sequences

[0503] Metallothionein IIA (J00271, V00594, X97260, S52379, P02795)

[0504] Interferon gamma (AF330164)

[0505] Platelet-derived growth factor B chain (PDGFB) (Y14326,XM_(—)009997)

[0506] Platelet-derived growth factor alpha polypeptide (PDGFA)(NM_(—)002607)

[0507] Neuregulin 1 (NRG1) (NM_(—)013964)

[0508] Heregulin-betal (M94166)

[0509] TNF-alpha (AB048818)

[0510] TNF-beta (Lymphotoxin) (D12614)

[0511] Oxytocin receptor (OXTR) (NM_(—)000916, X80282 M25650)

[0512] Kappa light chain nuclear factor, NFKB (L01459)

[0513] Selectin P (NM_(—)003005)

[0514] Selectin E (NM_(—)000450)

[0515] Integrin, alpha (NM_(—)000885)

[0516] Hormone-sensitive lipase (NM_(—)005357)

[0517] TGF-beta 1 (A18277)

[0518] ICAM-1 (X84737)

[0519] GM-CSF (AJ224149)

[0520] CD8 antigen (NM_(—)004931)

[0521] CD11A antigen, integrin alpha L (XM_(—)008099)

[0522] CD11b (NM_(—)000632)

[0523] CD11C (NM_(—)000887)

[0524] CD28 glycoprotein (AH002636)

[0525] CD34 antigen (CD34) (NM_(—)001773)

[0526] CD40 (XM_(—)009624)

[0527] CD40 ligand (X67878 S50586)

[0528] CD44 (NT_(—)024229)

[0529] CD54 (NT_(—)011130 NT_(—)004939)

[0530] CD58 (XM_(—)001325)

[0531] CD62L (NT_(—)004939)

[0532] CD69 antigen (BC007037)

[0533] CD80 antigen (CD28 antigen ligand 1, B7-1 antigen) (XM_(—)002948)

[0534] CD86 antigen (CD28 antigen ligand 2, B7-2 antigen) (XM_(—)002802)

[0535] Interleukin 1, beta (IL1B) (NM_(—)000576)

[0536] Interleukin 1 receptor antagonist (IL1-RA) (XM_(—)010756 P18510NM_(—)000577 AJ005835 BC009745 M55646 M63099 X52015 X53296 X64532 X84348AF043143)

[0537] Interleukin 2 (IL2) (AF359939)

[0538] Interleukin 2 receptor, beta (IL2R) (XM_(—)009962)

[0539] Interleukin 4 (IL4) (AF395008)

[0540] Interleukin 5 (IL5) (AF353265)

[0541] Interleukin 6 (IL6) (AF048692)

[0542] Interleukin 10 (IL10) (XM_(—)001409)

[0543] Interleukin 12A (NM_(—)000882)

[0544] Interleukin 12B (NM_(—)002187)

[0545] Interleukin 13 (IL13) (AF377331)

[0546] Interleukin 16 (NM_(—)004513)

[0547] Aldose reductase (BC010391)

[0548] Neutrophil elastase (AC004799)

[0549] Folate binding protein (FBP) (X62753)

[0550] Cytochrome c oxidase subunit Vb (Cox Vb) (M19961)

[0551] Cytochrome c oxidse subunit IV (Cox IV) (BC008704)

[0552] Transcription factor A, mitochondrial (TFAM) (NM_(—)012251)

[0553] ATP synthase beta (NM_(—)001686)

[0554] Prolactin (PRL) (XM_(—)004269)

[0555] Retinoic acid receptor, beta (RARB) (XM_(—)003071)

[0556] Choline acetyltransferase (CHAT) (XM_(—)011848)

[0557] Cholinergic receptor, nicotinic, beta polypeptide 4 (CHRNB4)(NM_(—)000750)

[0558] RAF1 (NM_(—)002880)

[0559] Nicotinic acetylcholine receptor (AChR) (X 17104)

[0560] Acetylcholine receptor delta subunit (X55019 X53091 X53516)

[0561] Cholinergic receptor, nicotinic, epsilon polypeptide(XM_(—)008520)

[0562] PKC alpha (X52479)

[0563] v-Ha-ras (XM_(—)006146)

[0564] v-fos FBJ murine osteosarcoma viral oncogene homolog (FOS)(NM_(—)005252)

[0565] Cytochrome P450 monoxygenase CYP2J2 (U37143)

[0566] Fibronectin (E01162)

[0567] Vascular cell adhesion molecule 1 (VCAM-1) (X53051)

[0568] PECAM1 (NM_(—)000442)

[0569] MCP-1 (Y18933)

[0570] AP-2 (X77343)

[0571] Apob-100 (M14162)

[0572] Actin, beta (ACTB) (XM_(—)004814)

[0573] GAPDH (NT_(—)009731)

[0574] Cyclin-dependent kinase 4 (CDK4) (NM_(—)000075)

[0575] Cyclin-dependent kinase 2 (CDK2) (XM_(—)006726)

[0576] Human cyclin D1 (M64349)

[0577] Human cyclin D2 (X68452)

[0578] Human cyclin A1 (NM_(—)003914)

[0579] Skeletal muscle alpha-actin (ACTA1) (AF182035)

[0580] Retinoic acid receptor, alpha (BC008727)

[0581] Transforming growth factor-beta (TGF-beta) (X02812 J05114)

[0582] Beta-1-adrenergic receptor (ADRB1) (AF169007)

[0583] Adrenergic, beta-2-, receptor, surface (ADRB2) (NM_(—)000024)

[0584] Insulin (BC005255)

[0585] Leptin (Lep) (U65742)

[0586] Leptin receptor db form (OB-Rdb) (U58863)

[0587] Myelin basic protein (MBP) (XM_(—)008797)

[0588] RANTES (AF088219)

[0589] MIP-1 alpha /RANTES receptor (E13385)

[0590] MIP-1 beta (NT_(—)010795)

[0591] Chemokine (C-C motif) receptor 5 (CCR5) (NM_(—)000579)

[0592] Thioredoxin (TXN) (XM_(—)015718)

[0593] Thrombopoietin (XM_(—)002815)

[0594] Polyomavirus (NC_(—)001515 NC_(—)001516)

[0595] JC virus (J02226 J02227 NC_(—)001699)

[0596] SV40 (J02400 J02402-3 J02406-10 J04139 M24874 M24914 M28728V01380 NC_(—)001669)

[0597] BK virus (NC_(—)001538 V01108 J02038 strain dunlop V01109 J02039strain MM J02038 K00058 V01108 strain dunlop M23122 strain AS)

[0598] Lymphotropic polyomavirus (K02562)

[0599] Human adenovirus type 2 (NC_(—)001405)

[0600] Human adenovirus 5 (NC_(—)001406 M73260 M29978)

[0601] Human adenovirus type 5 E1A enhancer (M13156)

[0602] Human adenovirus 17 (NC_(—)002067 AF108105)

[0603] Human adenovirus 40 (L19443)

[0604] Human herpesvirus 1 (NC_(—)001806 X14112 D00317 D00374 S40593)

[0605] Human herpesvirus 2 (NC_(—)001798)

[0606] Human herpesvirus 3 (NC_(—)001348)

[0607] Human herpesvirus 4 (NC_(—)001345)

[0608] Human herpesvirus 5 (NC_(—)001347 X04650 D00328 D00327 X17403(strain AD169) M17956 M21295 U33331 D63854 K01263 M60321 X03922 M1129M18921)

[0609] Human herpesvirus 6 (NC_(—)001664 X83413 (U1102, variant A)AB021506 (variant B, strain HST))

[0610] Human herpesvirus 6B (NC_(—)000898 AF157706 L13162 L14772 L16947(strain Z29))

[0611] Human herpesvirus 7 (NC_(—)001716 U43400 (JI) AF037218 (strainRK))

[0612] Epstein-Barr virus (EBV) (V01555 J02070 K01729-30 V01554X00498-99 X00784 (strain B95-8) L07923 X58140 D10059)

[0613] Rous sarcoma virus (NC_(—)001407)

[0614] Y73 sarcoma virus (NC_(—)001404)

[0615] Human coxsackievirus A (NC_(—)001429)

[0616] Coxsackievirus B3 (NC_(—)001473)

[0617] Moloney murine leukemia virus (NC_(—)001501 J02255 J02256 J02257M76668 AF033811)

[0618] Human immunodeficiency virus type 1 (AJ006022 NC_(—)001802 K02013K03455 M38432 AF286239 U86780 AF256211 AF256205 AF256207 AF256206 X04415K03456)

[0619] Human immunodeficiency virus type 2 (NC_(—)001722 J04542 U27200L14545 D00835 U38293 X05291 M31113 X52223 M15390 J04498 M30502 U22047L07625 M30895 D00477 X61240 X16109 AF082339)

[0620] Human T-cell lymphotropic virus type 1 (AF033817 NC_(—)001436AF259264 U19949 AF042071 J02029 M33896 AF139170 L03561)

[0621] Human T-cell lymphotropic virus type 2 (AF326584 NC_(—)001488AF326583 AF139382 Y13051 Y14365 AF074965 NC_(—)001877)

[0622] LCMV (Y16308 M20869 M22138 AF079517AF186080 AJ233196 AJ297484AJ233200 AJ233161 AH004719 AH004717 AH004715 S75753 S75741 S75739 912860912868)

[0623] TMEV (NC_(—)001366 AF030574 M80890 M80889 M80888 M80887 M80886M80885 M80884 M80883 M16020 M14703 M20562 M20301 M94868)

[0624] Hepatitis B virus (NC_(—)001707 AF330110 AB042283 AB042282AB050018 AB042284 AB049609 AB049610 AF182803 AB042285 AF182804 AF182805AF182802 AF384371 AF363961 AF384372)

[0625] Collagen type 1 alpha2 (COL1A2) (M35391 K02568 AF004877 AC002528M22817 M20904 XM_(—)004658 Z74616 L47668 NM_(—)000089 M22816 M20904J03464 Ml 8057 X02488 M21671 Y00724 V00503 S89896 M64229 S96821 AB004317L00613 U79752 S62614 S59218 S59211 S89898 X67667 P08123)

[0626] Collagen type I alpha 1 (COL1A1) (XM_(—)037910 AF017178)

[0627] Tissue factor (XM_(—)001322 J02931 J02681 NM_(—)001993 M16553J02846 M27436 AL138758 A19048 P13726 P30931 AAB20755 KFBO3 X53521 KFRB3P24055 AAA63469 CAA37597 AAF36523 Q9JLU8 M26071 AAA40414 KFMS3NP_(—)034301 P20352 AAA63400 AAA16966 P42533 NP_(—)037189)

[0628] Integrin, beta 2 (CD18) (X64074 X63835 X64075 X63835 X64076X63835 X64077 X63835 X64078 X63835 X64079 X63835 X64080 X63835 X64081X63835 X64082 X63835 X64083 X63835 X63924 X63835 X63925 X63835 X63926X63835 X64073 X63835 AL163300 AP001755 BA000005 BC005861 S81234 Y00057M19545 M15395 NM_(—)000211 X64071 X63835 X63926 X63835 AH003850 S81231S81252 S81247 S75381 S75297 M95293 M38701 X54481 M77675 P05107)

[0629] Rb1 (L11910 M27845 M27846 M27847 M27848 M27849 M27850 M27851L35146 M27852 M27853 M27854 M27855 M27856 M27857 M27858 M27859 M27860L35147 M27862 M27863 M27864 M27865 M27866 X16439 L41890 L41891 L41893L41894 L41895 L41896 L41897 L41898 L41899 L41997 L41999 L41907 L41914L41904 L41921 L41996 L41998 L42000 L41911 L41924 L41923 L41920 L41918L41870 L49209 L49212 L49213 L49218 L49220 L49223 L49230 L49231 L49232AH006304 AH005289 AH005290 AH005288 M26460 M28736 M15400 M28419 M33647J02994 NM_(—)000321 AF043224 XM_(—)007211 M19701 J03809 AAA53483)

[0630] BRCA1 (U37574 XM_(—)008213 XM_(—)008214 XM_(—)008215 XM_(—)008216XM_(—)008217 XM_(—)008219 XM_(—)008220 XM_(—)008221 XM_(—)008222XM_(—)017568 XM_(—)017569 XM_(—)017570 NM_(—)007294 NM_(—)007295NM_(—)007296 NM_(—)007297 NM_(—)007298 NM_(—)007299 NM_(—)007300NM_(—)007301 NM_(—)007302 NM_(—)007303 NM_(—)007304 NM_(—)007305NM_(—)007306 U14680 AF005068 U68041 U64805 Y08864 XP_(—)017569XP_(—)008212)

[0631] Fas (X63717 NM_(—)000043 X83493 X89101 Z47993 Z47994 Z47995Z70519 Z70520 P25445)

[0632] p300 (XM_(—)010013 U01877 NM_(—)001429 Q09472 S67605 AL096765)

[0633] CREB-binding protein (CBP) (AC004760 NP_(—)004371 AJ251844 U47741U85962 U89354 U89355 XM_(—)036668 XM_(—)036667 XM_(—)036669 BG₇₁₀₀₈₁S66385 U88570)

[0634] ZF_TAZ matrix, p300/cbp protein binding site (PS50134XM_(—)017011 XM_(—)009709 XM_(—)017011 AF078104 M74515 M74511 AF057717)

[0635] E4TF1-60 (D13318 X84366)

[0636] E4TF1-53 (D13317)

[0637] E4TF1-47 (D13316)

[0638] Human nuclear respiratory factor-2 subunit alpha (U13044)

[0639] Human nuclear respiratory factor-2 subunit beta 1 (U13045)

[0640] Human nuclear respiratory factor-2 subunit beta 2 (U13046)

[0641] Human nuclear respiratory factor-2 subunit gamma 2 (U13048)

[0642] GA-binding protein, subunit beta 1 (NM_(—)005254 NM_(—)016654BC004103 M74516 M74512)

[0643] GA-binding protein, subunit beta 2 (M_(—)002041 NM_(—)016655M74517 M74513)

[0644] GA-binding protein, subunit gamma 1 (U13047)

[0645] Ets1 (J04101 X14798 NM_(—)005238 M11921 XM_(—)015368XP_(—)015368)

[0646] ERK1 (AJ222708 NM_(—)002745 M84490 BC000205 Z11696 S38872 P27361Z11694 S38867 Z11695 S38869)

[0647] ERK2 (M84489 P28482)

[0648] JNK1 beta 2 (U35005)

[0649] JNK1 beta 1 (U35004)

[0650] JNK2 beta 2 (U35003)

[0651] JNK2 beta 1 (U35002)

[0652] JNK1 alpha 2 (U34822)

[0653] JNK2 alpha 1 (U34821)

[0654] JNK3 alpha 1 (U34820)

[0655] JNK3 alpha 2 (U34819)

[0656] JNK2 (L31951)

[0657] JNK1 beta 2 (AAC50611)

[0658] MEK1 (L05624 NM_(—)002755 Q02750)

[0659] MEK kinase 1 (MEKK1) (AF042838)

[0660] MEK kinase 3 (MEKK3) (U78876)

[0661] Human STAT1 (P42224 NM_(—)007315 AF182311 BC002704 M97936 U18662U18663 U18664 U18665 U18666 U18667 U18668 U18669 U18670)

[0662] Human STAT2 (U18671 M97934 S81491 P52630)

[0663] Human IL-2 receptor, gamma (NM_(—)000206 D11086 L12183 AC087668L19546 P31785)

[0664] Alpha 2 adrenergic receptor (M18415)

[0665] Beta 3 adrenergic receptor (P13945 X72861)

[0666] Beta 3 adrenergic receptorX70811)

[0667] Beta 3 adrenergic receptor (-X70812)

[0668] Beta 3 adrenergic receptor (S53291)

[0669] CCAAT/enhancer binding protein (C/EBP) (NM_(—)005194)

[0670] Cbp/p300-interacting transactivator (BC004240)

[0671] AML₁ (AF312387 AF025841 AF312386 AY004251)

[0672] AML (D10570)

[0673] AML1 (D43967 D43969 D89788 D89789 D89790 L21756 L34598 M83215U19601 X79549 X90976 X90978 X90981 AP001721 Q01196)

[0674] A-Myb (X66087 S75881 X13294 P10243)

[0675] ATF1 (X55544)

[0676] ATF2 (P15336 AY029364 M31630 U16028 X15875)

[0677] ATF4 (P18848 AL022312 BC008090 BC011994 D90209 M86842)

[0678] c-Fos (P01100 AB022276 AF111167 BC004490 K00650 V01512)

[0679] AP1 (P05412 AL136985 BC002646 BC006175 BC009874 J04111)

[0680] C2TA (P33076 AF410154 U18259 U18288 U31931 X74301)

[0681] c-Myb (P10242 AF104863 M13665 M13666 M15024 U22376 X52125 P17676AL161937 BC005132 BC007538 X52560 P16220 BC010636 M27691 M34356 S72459X555450

[0682] CREB (X60003 0431860

[0683] CRX (AF024711)

[0684] CID (P19538)

[0685] DBP (Q10586 BC011965 D28468 U06936 U48213 U792830

[0686] E2F1 (Q01094 AF086380 AL121906 BC005098 M96577 S49592 S74230U47675 U47677)

[0687] E2F2 (Q14209 AL021154 L22846)

[0688] E2F3 (000716 AL136303 D38550 Y10479)

[0689] Egr1 (P18146 AJ243425 M62829 M80583 X52541)

[0690] ELK1 (P19419 AB016193 AB016194 AF000672 AF080615 AF080616AL009172 M25269)

[0691] Ets2 (P15036 AF017257 AL163278 AP001732 J04102 M11922 X55181)

[0692] ER81 (P50549 AC004857 U17163 X87175 P03372 AF120105 AF172068AF172069 AF258449 AF258450 AF258451 AL078582 AL356311 M12674 S80316U476780

[0693] ER alpha (X03635 X624620

[0694] ER beta (Q92731 AB006589 AB006590 AF051427 AF051428 AF060555AF061054 AF061055 AF074598 AF074599 AF124790 AF215937 X99101)

[0695] GATA1 (P15976 AF196971 BC009797 M30601 X17254)

[0696] Gli3 (P10071 AC005028 AJ250408 M20674 M57609 P04150 AC005601BC015610 M109010

[0697] GR (M69104 M73816 U01351 U80946 X03225 X03348 Q16665 AF050127AF207601 AF207602 AF2084870

[0698] HIF1A (AF304431 BC012527 U22431 U29165 U85044 X72726)

[0699] HNF4A (P41235 AL132772 U72967 X76930 X87870 X87871 X87872 Z49825)

[0700] JunB (P17275 BC004250 BC009465 BC009466 M29039 U20734 X51345)

[0701] MDM2 (Q00987 AF201370 AF385322 AF385323 AF385324 AF385326AF385327 AJ276888 AJ278975 AJ278976 AJ278977 AJ278978 BC009893 M92424U33199 U33200 U33201 U33202 U33203 Z12020 NM_(—)006878 NM_(—)006879NM_(—)006880 NM_(—)006881 NM_(—)006882)

[0702] MDMD2 (AF385325)

[0703] MEF2C (Q06413 L08895 S57212)

[0704] Mi (O75030 AB006909 AB009608 AB032357 AB032358 AB032359 AL110195Z29678)

[0705] MyoD (P15172 AF027148 BC000353 X17650 X56677)

[0706] RelA (Q04206 BC011603 BC014095 L19067 M62399 Z22948 Z22951)

[0707] NFAT1 (Q13469 AL035682 U43341 U43342)

[0708] NF-YB (P25208 BC005316 BC005317 BC007035 L06145 X59710)

[0709] NF-YA (P23511 NM_(—)021705 AK025201 AL031778 M59079 X59711)

[0710] P/CAF (Q92831)

[0711] p/CIP (Q9Y6Q9 AL0344180 Q9UPG4)

[0712] MRG1 (Q99967 AF109161 AF129290 BC004377 U65093)

[0713] NFE2 (Q16621 BC005044 L13974 L24122 S77763 P04637 AF052180AF066082 AF135121 AF136271 AF307851 BC003596 K03199 M13121 M14694 M14695M22881 M22898 U94788 X01405 X02469 X541560 X60010 X600110

[0714] p53 (X60012 X60013 X60014 X60015 X60016 X60017 X60018 X60019X60020)

[0715] p73 (O15350 AF077628 AL136528 Y11416)

[0716] RSK1 (NM_(—)002953 AL109743 BC014966 L07597 Q15418)

[0717] RSK3 (AL022069 AX019387 BC002363 L07598 X85106)

[0718] RSK2 (P51812 L07599 U08316)

[0719] PIT1 (P28069 D10216 D12892 L18781 X62429 X72215)

[0720] RARG (P13631 AJ250835 L12060 M24857 M38258 M57707 P22932)

[0721] RXRA (AF052092 BC007925 BC009882 U66306 X52773 Q08211 L13848U03643 Y10658 P28324 NM 001973 M85164M85165 Q13285 D842060

[0722] SF-1(D84207 D84208 D842090 D84210 D88155 U76388 Q13485 AF0454470

[0723] SMAD4 (BC002379 U44378 Q15797 BC001878 U548260

[0724] SMAD1 (U57456 U59423 U59912)

[0725] SMAD2 (Q15796 AF027964 BC014840 U59911 U65019 U68018 U78733)

[0726] SMAD3 (Q92940 U68019 U76622)

[0727] SRC1 (AJ000882 NM_(—)0037430 AJ000881 U19177 U19179 U40396 U59302U90661)

[0728] SREBP1 (P36956 U00968)

[0729] SREBP2 (Q12772 U02031 Z99716)

[0730] STAT3 (P40763 AJ012463 BC000627 BC014482 L29277)

[0731] STAT4 (Q14765)

[0732] STAT5A (P42229 L41142 U43185)

[0733] STAT5B (P51692 U47686 U48730 P42226 AF067572 AF067573 AF067574AF067575 BC004973 BC0058230

[0734] STAT6 (U16031 U66574)

[0735] TAL1 (P17542 AJ131016 AL135960 M29038 M61108 S53245 X51990)

[0736] TBP (P20226 AL031259 M34960 M55654 X54993)

[0737] TF2B (Q00403 AL445991 S44184)

[0738] THRA (P10827 BC000261 BC002728 J03239 M24748 M24899 X55005 X55074Y00479)

[0739] THRB (P10828 M26747 X04707 P37243)

[0740] TWIST (Q15672 U80998 X91662 X99268 Y10871)

[0741] IRF3 (Q14653 AF112181 AX015330 AX015339 BC009395 U86636 Z56281)

[0742] YY1 (P25490 AF047455 M76541 M77698 Z14077)

[0743] PPARG (P37231 NM_(—)015869 BC006811 D83233 L40904 U63415 U79012X90563)

[0744] AR (P10275 AF162704 L29496 M20132 M20260 M21748 M23263 M27430M34233 M35851 M58158 S79366 S79368 M27424 M27425 M27426 M27427 M27428M27429 M35845 M35846 M35847 M35848 M35849 M35850)

[0745] SRD5A1 (P18405 AF052126 AF113128 AL008713 BC006373 BC007033BC008673 M32313 M68886 M68882 M68883 M68884 M68885 AF073302 AF073304)

[0746] (b) Equivalent Molecules

[0747] The term “equivalent molecules” is understood to includemolecules having the same or similar activity as the molecule ofinterest, including, but not limited to, biological activity andchemical activity, in vitro or in vivo.

[0748] (c) Homologous Molecules

[0749] The term “homologous molecules” is understood to includemolecules with the same or similar chemical structure as the molecule ofinterest (see exemplary embodiments above).

[0750] The following section presents standard assays which can be used,in conjunction with the assays in the new elements section, to test theeffect of an agent on a molecule of interest.

[0751] (d) During

[0752] The term “during drug discovery, development, use as treatment,or during diagnosis” is understood to include, but not be limited to,drug screening, rational design, optimization, in laboratory or clinicaltrials, in vitro or in vivo (see exemplary embodiment below).

[0753] (2) Assaying Protein Concentration

[0754] (a) UV Absorbance

[0755] In one exemplary embodiment, cellular protein concentration ismeasured by virtue of its absorbance of ultraviolet light at thewavelength of 280 nm (Ausubel 1999¹⁵⁵). To calibrate the reagents used,and to validate the spectrophotometer, a standard curve is establishedusing protein solutions of known concentration. Typically solutions ofbovine serum albumin, a commonly available protein, are used toestablish the standard curve. Cells are lysed in a detergent-rich bufferto liberate membrane associated and intracellular proteins. Followinglysis, insoluble materials are removed by centrifugation. The absorbanceof UV light by the supernatant, which contains soluble proteins ofunknown concentration, is then measured and compared to the standardcurve. Comparison of the data obtained from the cellular extracts withthose represented by the standard curve provides an indication ofcellular protein concentration.

[0756] (b) Bradford Method

[0757] In another exemplary embodiment, protein concentration isdetermined using the Bradford method (Sapan 1999¹⁵⁶, Ausubel 1999,Ibid). A standard curve is constructed using solutions of known proteinconcentration mixed with coomassie brilliant blue. Following a briefincubation at room temperature, the absorbance of light at 595 nm ismeasured and a standard curve is constructed. Cells are lysed asdescribed above, the lysate is mixed with coomassie brilliant blue andthe absorbance measured in a manner identical to that of the standardcurve. Comparison of the values obtained from the cellular extract withthose of the solutions of known concentration reveals the concentrationof cellular proteins.

[0758] (c) Immunoaffinity Chromatography

[0759] To measure concentration of a specific cellular protein, forinstance, p300, GABP or CBP, additional steps are employed to purify theprotein away from other cellular proteins. One exemplary embodimentinvolves the use of specific antibodies targeted against the protein ofinterest to remove it from the cellular lysate. Specific antibodies, forinstance, anti-p300, anti-GABP or anti-CBP, are chemically bound to aresin and contained within a vertical glass or plastic column. Celllysate is passed over that resin to permit antibody-antigeninteractions, thereby allowing the protein to bind to the immobilizedantibodies. Efficient removal of the protein of interest from the celllysate is accomplished by using an excess of antibody. Protein bound tothe column is removed which releases the bound protein. The elutedprotein is collected and its concentration determined by an assay forprotein concentration such as those exemplified above.

[0760] (3) Assaying mRNA Concentration

[0761] (a) UV Absorbance

[0762] In certain embodiments, RNA concentration is measured byabsorption of ultraviolet light at a wavelength of 260 nm (Manchester1995¹⁵⁷, Davis 1986¹⁵⁸, Ausubel 1999, Ibid). RNA is purified from cellsby first lysing the cells in a detergent rich buffer. Proteins in thecellular lysate are degraded by incubation overnight at 65° C. withproteinase K. After enzymatic degradation, proteins are extracted fromthe solution by mixing with phenol/chloroform/isoamyl alcohol followedby extraction with chloroform/isoamyl alcohol. Nucleic acids in theresulting protein deficient solution are precipitated by addition ofsalt, typically sodium acetate or ammonium acetate, and ethanol. After abrief incubation of the mixture at −20° C., the insoluble nucleic acidsare removed by centrifugation, dried, and redissolved in a sterile,RNase free solution of Tris and EDTA. Contaminating DNA is removed fromthe lysate by treatment with RNase-free DNase I. Degraded DNA is removedby precipitation of the intact RNA with salt and ethanol. The dried,purified RNA is dissolved in Tris-EDTA and quantified by virtue of itsabsorbance of light at 260 nm. Since the molar extinction coefficient ofRNA at 260 nm is well known, the concentration of RNA in the solutioncan be determined directly.

[0763] (b) Northern Blot

[0764] The concentration of a particular RNA species can also bedetermined. In one exemplary embodiment, the amount of mRNA whichencodes a protein of interest, for instance, p300, GABP, CBP, within apopulation of cells is measured by Northern blot analysis (Ausubel 1999,Ibid, Gizard 2001¹⁵⁹). Total cellular RNA is isolated and separated byelectrophoresis through agarose under denaturing conditions, typicallyin a gel containing formaldehyde. The RNA is then transferred to, andimmobilized upon a charged nylon membrane. The membrane is incubatedwith a solution of detergent and excess of low molecular weight DNA,typically isolated from salmon sperm, to prevent adventitious binding ofthe gene specific, for instance, p300-, GABP-, CBP-specific,radiolabeled DNA probe to the membrane. Radiolabeled cDNA probesrepresenting the protein, e.g., p300, GABP, CBP, transcript are thenhybridized to the membranes and bound probe is visualized byautoradiography.

[0765] (c) Reverse Transcriptase—Polymerase Chain Reaction (RT-PCR)

[0766] In another exemplary embodiment, the amount of mRNA encoding aprotein of interest, for instance, p300, GABP, CBP, expressed by apopulation of cells is measured by first isolating RNA from cells andpreparing cDNA by binding oligo deoxythymidine (dT) to thepolyadenylated mRNA within the prepared RNA. Reverse transcriptase isthen used to extend the bound oligo dT primers in the presence of allfour deoxynucleotides to create DNA copies of the mRNA. The cDNApopulation is then amplified by the polymerase chain reaction in thepresence of oligonucleotide primers specific for the sequence of thegene or RNA of interest and Taq DNA polymerase. The amplificationproducts can be visualized by gel electrophoresis followed by stainingwith ethidium bromide and exposure to ultraviolet light. Quantificationcan be achieved by adding a radiolabeled deoxynucleotide to the PCRreaction. Radiolabel incorporated into the amplification products isvisualized by autoradiography and quantified by densitometric analysisof the autoradiograph or by direct phosphorimager analysis of theelectrophoretic gel.

[0767] (d) S1 Nuclease Protection

[0768] In a related exemplary embodiment, expression of RNA encoding aprotein of interest, for instance, p300, GABP, CBP, can be assessed byhybridizing isolated cellular RNA with a radiolableled synthetic DNAsequence homologous to the 5′ terminus of the RNA of the protein ofinterest. The synthetic deoxyribonucleotide, less than 40 nucleotides inlength, is labeled at it 5′ end with T4 polynucleotide kinase and γ-³²PATP. Once the oligonucleotide is bound to the RNA, the mixture isincubated in the presence of the single strand-specific nuclease S1. Anyunhybridized, and therefore single stranded, molecules of RNA or DNA aredegraded, leaving the DNA-RNA hybrids of the protein of interest intact.The undegraded hybrids are removed from the solution by precipitationwith ammonium acetate and ethanol and resolved by nondenaturing gelelectrophoresis. Radiolabeled bands on the gel are then visualized byautoradiography. The radiolabel can be quantified by densitometricanalysis of the autoradiographs or by phosphorimager analysis of theelectrophoretic gels themselves.

[0769] (4) Assaying Polynucleotide Copy Number

[0770] (a) S1 Nuclease Protection

[0771] This same technique can be used to quantify the level of anynucleic acid, naturally expressed or exogenous, within a population ofcells. In every case the sequence of the single stranded syntheticoligonucleotide must be designed so that it is complementary to the 5′terminal sequence of the species to be measured.

[0772] (b) Real Time PCR

[0773] In another exemplary embodiment, DNA copy number can be measuredusing real time PCR (Heid 1996¹⁶⁰). This technique employsoligonucleotides doubly labeled. At the 5′ ends they carry a reporterdye that fluoresces upon excitation by the appropriate wavelength oflight. At the 3′ end they carry a quencher dye that suppresses thefluorescence of the first dye. These oligonucleotides are prepared sothat their sequence is complementary to the region of interest whichlies between the forward and reverse PCR primers. Once hybridized to theDNA sequence of interest, the close proximity of the quencher dye andthe fluorescent dye suppresses the fluorescent emissions of the reporterdye. However, during the process of PCR, Taq polymerase cleaves thereporter dye from the oligonucleotide and releases it. Once removed fromthe nearby quencher dye, fluorescence is permitted. Free fluorescent dyeis quantified with a fluorimeter and is directly related to the numberof molecules of interest present prior to PCR.

[0774] (5) Detection of Binding

[0775] (a) General

[0776] In one exemplary embodiment, an assay to identify compounds thatbind to a polynucleotide or polypeptide of interest involves binding ofa test compound to wells of a microtiter plate by covalent ornon-covalent binding. For instance, the assay may anchor a specific testcompound to a microtiter plate substrate using a mono or polyclonalimmobilized antibody. A solution of the test compound can also be usedto coated the solid surface. Then, the nonimmobilized polynucleotide orpolypeptide of interest may be added to the surface coated wells. Aftersufficient time is allowed for the reaction to complete, the residualcomponents are removed by, for instance, washing. Care should be takennot to remove complexes anchored on the solid surface. Anchoredcomplexes may be detected by several methods known in the art. Forinstance, if the nonimmobilized polynucleotide or polypeptide ofinterest, or test compound were labeled before the reaction, the labelmay be used to detect the anchored complexes. If the components were notprelabeled, a label may be added during or after complex formation, forinstance, an antibody directed against the nonimmobilized polynucleotideor polypeptide of interest, or test compound, can be added to thesurface coated wells.

[0777] In a variation of this assay, the polynucleotide or polypeptideof interest is anchored to the a solid surface and the nonimmobilizedtest compound is added to the surface coated wells.

[0778] In another variation of this assay, the reactions are performedin a liquid phase, and the complexes are removed from the reactionmixture by immunoaffinity chromatography, or immunoprecipitation, asdescribed herein.

[0779] (b) Detection of Binding to DNA

[0780] In one exemplary embodiment, DNA fragments carrying a known, orsuspected binding domain for a polypeptide of interest, for instance,p300, GABP, etc., are purified by gel electrophoresis and labeled withT4 polynucleotide kinase in the presence of γ³²P-ATP (Bulman et al.2001). Labeled DNA is then added to a solution containing thepolypeptide of interest under conditions, ionic and thermal, whichpermit formation of DNA-polypeptide complexes. The solution is thenmaintained for a period of time sufficient for the reaction to complete.Following completion, the mixture is separated by electrophoresisthrough nondenaturing polyacrylamide in parallel to labeled, butotherwise unreacted test DNA. Following electrophoresis, the labeled DNAis detected by autoradiography or by phosphorimager analysis. Formationof complexes is detected by the shift in electrophoretic mobility (seealso below).

[0781] The assay detects polypeptide-DNA complexes formed by directbinding of the polypeptide of interest with DNA, or by indirect bindingthrough intermediary polypeptides, as long as the intermediarypolypeptides are present in the reaction mixture. Further, the magnitudeof the gel shift provides a semi-quantitative measure of the relativeconcentration of the polypeptide-DNA binding in the assay mixture. Assuch, changes in concentration can also be detected.

[0782] (i) Affinity Chromatography

[0783] In one exemplary embodiment, binding of a polypeptide ofinterest, that is, disrupted polypeptide, or polypeptide in a disruptedor disruptive pathway, such as p300, GABP, CBP, to DNA is measured byfirst expressing fragments of the polypeptide of interest as GST(glutathione sulfonyl transferase) fusion proteins in E. coli (Gizard2001, Ibid). The expressed polypeptides are then bound to glutathionecoupled sepharose. Radiolabeled DNA fragments, carrying ³²P,representing the polypeptide binding site, are incubated withprotein-bead complexes and subsequently washed three times to removeadventitiously bound DNA. Any DNA bound to the immobilized polypeptideof interest are released by boiling in presence of the ionic detergentSDS. Liberated radiolabeled DNA is quantified by liquid scintillationcounting, or by direct measurement of Cerenkov radiation.

[0784] (ii) Electrophoretic Gel Mobility Shift Assay

[0785] In another exemplary embodiment, binding of a polypeptide ofinterest, or a group of polypeptides to DNA is assessed byelectrophoretic gel mobility shift assay (Gizard 2001, Ibid, Ausubel1999, Ibid, Nuchprayoon 1999¹⁶¹). Radiolabeled DNA carrying thepolypeptide binding site, for instance, the p300 binding site, or N-box,is mixed with the recombinant polypeptide, for instance, p300, GABP,expressed as GST fusion protein. Subsequent resolution byelectrophoresis through nondenaturing polyacrylamide gels in parallelwith labeled DNA alone, reveals a shift in electrophoretic mobility onlyif the polypeptide is bound to DNA in the DNA/polypeptide mixtures. Ifthe DNA binding site is unknown, or one is suspected to be carried in acollection of DNA fragments, this assay can be performed to test for,and potentially affirm the presence of such a binding site.

[0786] (6) Detection of Binding Interference

[0787] A polynucleotide or polypeptide of interest may bind with one ormany cellular or extracellular proteins in vivo. Compounds thatinterfere with, or disrupt the binding may include, but are not limitedto, antisense oligonucleotides, antibodies, peptides, and similarmolecules.

[0788] In one exemplary embodiment, binding interference of a testcompound is assessed by adding the compound to a mixture containing apolynucleotide or polypeptide of interest and a binding partner. Afterenough time is allowed for the reaction to be completed, the complexconcentration in the test reaction mixture is compared to a controlmixture prepared without the test compound, or with a placebo. Adecreased concentration in the test reaction indicates interference.Reactants may be added at different orders regardless of the methodused. For example, a test compound may be added to the reaction mixturebefore adding the polynucleotide or polypeptide of interest and theirbinding partners, or at the same time. A test compound that can disruptan already formed complex, for instance, by displacing a complexcomponent, can be added to the reaction mixture after complex formation.The interference assay can be conducted in two ways, in liquid, or insolid phases, as described above.

[0789] In another embodiment, a polynucleotide or polypeptide ofinterest is prepared for immobilization by fusion toglutathione-S-transferase (GST), while maintaining the binding capacityof the fusion protein. Another complex component, a cellularpolynucleotide or polypeptide, or extracellular protein, can bepurified, and then utilized in developing a monoclonal antibody usingmethods well known in the art. The GST-polynucleotide fusion protein iscoupled to glutathione-agarose beads and exposed to the other complexcomponent in the presence or absence of a test compound. Aftersufficient time has been allowed for the reaction to complete, unboundcomponents are removed, for instance, by washing, and the labeledmonoclonal antibody is added. Bound radiolabeled antibody is thenmeasured to quantify the extent of complex formation. Inhibition ofcomplex formation by a test compound decreases measured radioactivity.As above, a test compound capable of complex disruption can also beadded after complex formation.

[0790] In one variation of the assay, the fusion protein is mixed withthe other complex component in liquid, that is, without solidglutathione-agarose beads.

[0791] In another variation of the assay, peptide fragments of thebinding domains, instead of full length complex components are used.Several methods well known in the art can be used to identify andisolate binding domains. For instance, one method entails mutating agene and screening for a disruption in normal binding of the polypeptideencoded by the gene by co-immunoprecipitation or immunoaffinity. If thepolypeptide shows disrupted binding, analysis of the gene sequence canreveal the binding domain, or the region of the polypeptide involved inbinding. Another approach partially proteolyzes a labeled polypeptideanchored to a solid surface. Non bound fragments are removed by washingleaving a labeled polypeptide comprising the binding domain immobilizedon the solid surface. The polypeptide fragments bound to the immobilizedproteins are than isolated and analyzed by amino acid sequencing, usingfor instance the Edman degradation procedure (Creighton 1983¹⁶²).Another approach expresses specific fragments of a polynucleotide, orgene, and tests the fragments for binding activity.

[0792] In another embodiment, an assay uses a complex with one componentlabeled. However, binding to the complex quenches the signal generatedby the label (see, for instance, U.S. Pat. No. 4,109,496). A testcompound which disrupts the complex, for instance, by displacing a partof the complex, restores the signal. This assay can be used to identifycompounds which either interfere with complex formation, or disrupt analready formed complex.

[0793] Specifically, a test compound can interfere with binding betweena disrupted gene or polypeptide, or a gene or polypeptide in adisruptive or disrupted pathway, for instance, a microcompeted ormutated gene or polypeptide, and their binding partner. The assay may beespecially useful in identifying compounds capable of interfering inbinding reactions between foreign polynucleotides and cellularpolypeptides without interfering in binding between cellularpolynucleotide and cellular polypeptides. The assay is also especiallyuseful in identifying compounds capable of interfering in bindingbetween mutant cellular polynucleotide or polypeptide and normalcellular polynucleotide or polypeptide without interfering in bindingbetween normal polynucleotide or polypeptides.

[0794] (7) Identification of a Polypeptide Bound to DNA or ProteinComplex

[0795] (a) Immunoprecipitation

[0796] In one exemplary embodiments, the identity of a boundpolypeptide, for instance, p300, GABP, CBP, is confirmed by reactingantibodies specific to the polypeptide of interest with polypeptidesbound to DNA. For example, p300-specific antibodies are mixed with thepolypeptide-DNA complexes and incubated overnight at 4° C. Immunecomplexes are then precipitated by the addition of a secondary antibodydirected against the primary p300-specific antibody. Precipitatedantibody-antigen complexes are resolved by denaturing gelelectrophoresis and the constituent proteins are visualized by stainingwith coomassie brilliant blue.

[0797] In a related exemplary embodiment, the interaction between apolypeptide of interest, for instance, p300, GABP, CBP, and othercellular proteins, such as transcription factors, may be detected byco-immunoprecipitation of the polypeptide of interest with antibodiesspecific to the polypeptide, for instance, p300-specific antibodies. Forexample, in the case of p300, cellular protein extracts are incubatedwith purified p300-GST fusion proteins to enable protein-proteininteractions. p300-specific antibodies are then added and the mixture isincubated overnight at 4° C. Immune complexes are precipitated byaddition of a secondary antibody directed against the primary p300antibodies and the precipitates are resolved by electrophoresis ondenaturing polyacrylamide gels. Proteins are subsequently detected bystaining with coomassie brilliant blue.

[0798] (b) Antibody Supershift Assay

[0799] In a related exemplary embodiment, DNA-protein complexes aredetected by electrophoretic gel mobility shift assay (Gizard 2001, Ibid,Ausubel 1999, Ibid). Radiolabeled DNA carrying the polypeptide bindingsite, for instance, p300 binding site, or N-box, is mixed with arecombinant polypeptide, for instance, p300, or GABP, expressed as GSTfusion protein. Subsequent resolution by electrophoresis throughnondenaturing polyacrylamide gels in parallel with labeled DNA alone,reveals a shift in electrophoretic mobility only if the polypeptide isbound to DNA in the DNA/polypeptide mixture. To identify the boundpolypeptide, a specific antibody is reacted to the DNA/polypeptidemixture prior to electrophoresis. Bound antibody molecules cause afurther change in gel mobility, namely a supershift, and serve toidentify the polypeptide bound to DNA.

[0800] (8) Identification of a DNA Consensus Binding Site

[0801] (a) PCR and DNA Sequencing

[0802] In one exemplary embodiment, DNA fragments are preparedcontaining potential polypeptide binding sites, either wild-type orvariants, flanked by DNA fragments of known nucleotide sequence. Thefragments are then reacted with the polypeptide-GST fusion proteinsimmobilized on sepharose beads. After washing to remove adventitiouslybound DNA, bound fragments are eluted by heating in presence of adetergent. The eluted fragments are amplified by the polymerase chainreaction (PCR) using primers specific for the flanking DNA sequences.The nucleotide sequence of the amplification products is then determinedby any sequencing method known in the art, for instance, the dideoxychain termination sequencing method of Sanger (Sanger 1977¹⁶³), using assequencing primer one of the two PCR primers. Several sequence variantsof the binding site are likely to be identified. Together they can beused to establish a consensus DNA sequence for the polypeptide bindingsite.

[0803] (9) Detection of a Genetic Lesion

[0804] Existence of a genetic lesion can be determined by observing oneor more of the following irregularities.

[0805] 1. Deletion of at least one nucleotide from a disrupted gene, orgene in a disrupted pathway.

[0806] 2. Addition of at least one nucleotide to a disrupted gene, or agene in a disrupted pathway.

[0807] 3. Substitution of at least one nucleotide to a disrupted gene,or gene in a disrupted pathway.

[0808] 4. Irregular modification of a disrupted gene, or gene in adisrupted pathway, such as change in DNA methylation patterns.

[0809] 5. Gross chromosomal rearrangement of a disrupted gene, or genein a disrupted pathway, for instance, translocation.

[0810] 6. Allelic loss of disrupted gene, or gene in a disruptedpathway.

[0811] 7. Different than wild-type mRNA concentration of a disruptedgene, or gene in a disrupted pathway.

[0812] 8. Irregular splicing pattern of mRNA transcript of a disruptedgene, or gene in a disrupted pathway.

[0813] 9. Irregular post-transcriptional modification of an mRNAtranscript other than splicing, for instance, editing, capping orpolyadenylation, of a disrupted gene or gene in a disrupted pathway.

[0814] 10. Different than wild-type concentration of a disruptedpolypeptide, or polypeptide in a disrupted pathway.

[0815] 11. Irregular post-translational modification of a disruptedpolypeptide, or a polypeptide in a disrupted pathway.

[0816] Many assays are known in the art for detection of the above, orother irregularities associated with a genetic lesion. Consider thefollowing exemplary assays. Also consider the exemplary assays discussedin the following reviews on detection of genetic lesions, Kristensen2001¹⁶⁴, Tawata 2000¹⁶⁵, Pecheniuk 2000¹⁶⁶, Cotton 199¹⁶⁷, Prosser1993¹⁶⁸, Abrams 1990¹⁶⁹, Forrest 1990¹⁷⁰.

[0817] (a) Sequencing

[0818] In one exemplary embodiment, a polynucleotide of interest can besequenced using any sequencing techniques known in the art to reveal alesion by comparing the test sequence to wild-type control, known mutantsequence, or sequences available in public databases.

[0819] An introduction to sequencing is available in Graham 2001¹⁷¹.Exemplary sequencing protocols are available in Rapley 1996¹⁷². Recentsequencing methods are available in Marziali 2001¹⁷³, Dovichi 2001¹⁷⁴,Huang 1999¹⁷⁵, Schmalzing 1999¹⁷⁶, Murray 1996¹⁷⁷, Cohen 1996¹⁷⁸,Griffin 1993¹⁷⁹. Automated sequencing methods are available in Watts2001¹⁸⁰, MacBeath 2001¹⁸¹, Smith 1996¹⁸². For classical sequencingmethods see Maxam 1977¹⁸³, Sanger 1977 (Ibid).

[0820] (b) Restriction Enzyme Cleavage Patterns

[0821] In another exemplary embodiment, patterns of restriction enzymecleavage are analyzed to reveal lesions in a polynucleotide of interest.For example, sample and control DNA are isolated, amplified, ifnecessary, digested with one or several restriction endonucleases, andthe fragments separated by gel electrophoresis. Sequence specificribozymes are then used to detect specific mutations by development orloss of a ribozyme cleavage site.

[0822] (c) Protection From Cleavage Agents

[0823] In another exemplary embodiment, cleavage agents, such as certainsingle-strand specific nucleases, hydroxylamine, osmium tetroxide orpiperidine, are used to detect mismatched base pairs in nucleic acidhybrids comprised of either RNA/RNA or RNA/DNA duplexes. Wild-type andtest DNA or RNA, with one or the other molecule labeled withradioactivity, are mixed under conditions permitting formation ofheteroduplexes between the two species. Following hybridization, theduplexes formed are treated with an agent capable of cleaving single,but not double stranded nucleic acids. Examples include, but are notlimited to S1 nuclease, piperidine, hydroxylamine and RNase H, in thecase of RNA/DNA heteroduplexes. Since mismatches between wild-type andmutant oligonucleotide result in single stranded regions, mismatch sitesare susceptible to digestion. Once cleaved, the nucleic acid fragmentsare separated according to size by native polyacrylamide gelelectrophoresis. Genetic lesion are detected by, for instance, observingdifferent fragment sizes in test relative to wild-type DNA or RNA.

[0824] Examples of such assay in practice are available in Saleeba1992¹⁸⁴, Takahashi 1990¹⁸⁵, Cotton 1988¹⁸⁶, Myers 1985A¹⁸⁷, Myers1985B¹⁸⁸.

[0825] (d) Mismatched Base Pairs Recognition

[0826] In another exemplary embodiment, mismatch cleavage reactions arecarried out using one or more proteins capable of recognizing mismatchedbase pairs. The proteins are typically components of the naturallyoccurring DNA mismatch repair mechanism. In a preferred embodiment, themutY enzyme derived from E. coli cleaves the adenine at a G/A mismatch(Xu 1996¹⁸⁹). The enzyme thymidine DNA glycosylase, isolated from thehuman cell line HeLa, cleaves the thymidine at G/T mismatches (Hsu1994¹⁹⁰). In practice, a probe is used comprising the wild-type sequenceof interest. The probe is hybridized to DNA, or cDNA corresponding tomRNA of interest. Once duplex formation has reached completion, a DNAmismatch repair enzyme is added to the reaction, and the products of thecleavage are detected by, for instance, separating reactants bydenaturing polyacrylamide gel electrophoresis.

[0827] (e) Alterations in Electrophoretic Mobility

[0828] In another exemplary embodiment, variations in electrophoreticmobility are used to identify genetic lesions, by standard techniques,such as single strand conformation polymorphism (SSCP) (Miterski2000¹⁹¹, Jaeckel 1998¹⁹², Cotton 1993, Ibid, Hayashi 1992¹⁹³). Dilutepreparations of radiolabeled single-stranded DNA fragments of test andcontrol nucleic acids, separately, are denatured by heat and permittedto renature slowly. Upon renaturation, single stranded nucleic acids inthe dilute solutions form secondary structures. Each molecule formsinternal base paired regions depending on each molecule sequence.Consequently, wild-type and mutant sequences, otherwise identical exceptfor regions of mutation, form different secondary structures. Eachpreparation is separated in adjacent lanes by electrophoresis throughnative polyacrylamide gels while preserving the secondary structureformed during renaturation. Alterations in electrophoretic mobilityreveal differences between wild-type and mutant oligonucleotides assmall as single nucleotide differences. Following electrophoresis theradiolabeled nucleic acids are detected by autoradiography or byphosphorimager analysis. A variation of this assay employs RNA ratherthan DNA.

[0829] In a related exemplary embodiment, wild-type and mutant DNAmolecules are separated by electrophoresis through polyacrylamide gelscontaining a gradient of denaturant. The method, termed “denaturinggradient gel electrophoresis,” (DGGE) (Myers 1985B, Ibid) is commonlyused to detect differences between similar oligonucleotides. Prior toanalysis, test DNA is often modified by addition of up to 40 base pairsof GC rich DNA through PCR. The relatively stable region, termed “GCclamp,” ensures only partial denaturation. A variation of the assayemploys a temperature rather than chemical gradient of denaturant.

[0830] (f) Selective Oligonucleotide Hybridization

[0831] In another embodiment, selective hybridization involves the useof synthetic oligonucleotide primers prepared to carry a known mutationin a central position. Primers are then mixed with test DNA underconditions permitting hybridization for perfectly matched molecules(Lipshutz 1995¹⁹⁴, Guo 1994¹⁹⁵, Saiki 1989¹⁹⁶). The allele specificoligonucleotide (ASO) hybridization method can be used to test a singlemutation per reaction mixture, or many different mutations if the ASO isfirst immobilized on a suitable membrane. The technique, termed “dotblotting,” permits rapid screening of many mutations when nonimmobilizedDNA is first radiolabeled to permit visualization of the immobilizedhybrids.

[0832] (g) Allele Specific Amplification

[0833] Under certain conditions, polymerase extension occurs only ifthere is a perfect match between primer and the 3′ terminus of the 5′,left-most or upstream region of a sequence of interest. Therefore, inanother embodiment, allele specific amplification, a selective PCRamplification based assay, a synthetic oligonucleotide primer isprepared carrying a mutation at the center, or extreme 3′ end of theprimer, such that mismatch between primer and test DNA prevents, orreduces efficiency of the polymerase extension during amplification(Efremov 1991¹⁹⁷, Gibbs 1989¹⁹⁸). A mutation in the test DNA is detectedby a change in amplification product concentration relative to controls,or, in special cases, by the presence or absence of amplificationproducts.

[0834] A variation of the assay introduces a novel restrictionendonuclease recognition site in the expected mutation region to permitdetection by restriction endonuclease cleavage of the amplificationproducts (see also above).

[0835] (h) Protein Truncation Test

[0836] Another embodiment uses the protein truncation test (PTT). If amutation introduces a premature translation stop site, PTT offers aneffective detection assay Geisler 2001¹⁹⁹, Moore 2000²⁰⁰, van der Luijt1994²⁰¹, Roest 1993²⁰²). In this assay, RNA is isolated from samplecells or tissue and converted to cDNA by reverse transcriptase. Thesequence of interest is amplified by the PCR, and the products aresubjected to another round of amplification with a primer carrying apromoter for RNA polymerase, a sequence for translation initiation. Theproducts of the second round of PCR are subjected to transcription andtranslation in vitro. Electrophoresis of the expressed polypeptidesthrough sodium dodecyl sulfate (SDS) containing polyacrylamide gelsreveals the presence of truncated species arising from the presence ofpremature translation stop sites. In a variation of this assay, if thesequence of interest is contained within a single exon, DNA rather thancDNA can used as PCR amplification template.

[0837] (i) General Comments

[0838] Any tissue or cell type expressing a sequence of interest may beused in the described assays. For instance, bodily fluids, such as bloodobtained by venipuncture or saliva, or non-fluid samples, such as hair,or skin, may be used. Samples of fetal polynucleotides collected frommaternal blood, amniocytes derived from amniocentesis, or chorionicvilli obtained for prenatal testing, can also be used.

[0839] Pre-packaged diagnostic kits containing one or more nucleic acidprobes, primer set, and antibody reagent may be useful in performing theassays. Such kits are designed to provide an easy to use instrumentespecially suitable for use in the clinic.

[0840] The assays may also be applied in situ directly on the tissue tobe tested, fixed or frozen. Typically, such tissue is obtained inbiopsies, or surgical procedures. In situ analysis precludes the needfor nucleic acid purification.

[0841] While the exemplary assays described so far primarily permit theanalysis of one nucleic acid sequence of interest, they may be also usedto generate a profile of multiple sequences of interest. The profile maybe generated, for example, by employing Northern blot analysis, adifferential display procedure, or reverse transcriptase-PCR (RT-PCR).

[0842] In addition to nucleic acid assays, antibodies directed against amutated polynucleotide, or polypeptide product of a mutatedpolynucleotide may be used in various assays (see below).

[0843] (10) Assaying Methylation Status of DNA

[0844] (a) Sodium Bisulfite Method

[0845] In one exemplary embodiment, the methylation status of DNAsequences can be determined by first isolating cellular DNA, and thenconverting unmethylated cytosines into uracil by treatment with sodiumbisulfite, leaving methylated cytosines unchanged. Following treatment,the bisulfite is removed, and the chemically treated DNA is used as atemplate for PCR. Two parallel PCR reactions are performed for each DNAsample, one using primers specific for the DNA prior to bisulfitetreatment, and one using primers for the chemically modified DNA. Theamplification products are resolved on native polyacrylamide gels andvisualized by staining with ethidium bromide followed by UVillumination. Amplification products detected from the sodium bisulfitetreated samples indicate methylation of the original sample.

[0846] Specifically, this assay can be used to asses the methylationstatus of DNA binding sites of a polypeptide of interest, such as GABP,p300, CBP, etc.

[0847] (11) Assaying Protein Phosphorylation

[0848] (a) Western Blot With Antiphosphotyrosine

[0849] In one exemplary embodiment, protein phosphorylation is measuredusing anti-phosphotyrosine antibodies (for instance, antibodiesavailable from Santa Cruz Biotechnology, catalog numbers sc-508 orsc-7020). Cultured cells are lysed by boiling in detergent-containingbuffer. Proteins contained in the cell lysate are separated byelectrophoresis through SDS polyacrylamide gels followed by transfer toa nylon membrane by electrophoresis, a process termed electroblotting(Burnett 1981²⁰³). Prior to incubation with antibody, the membrane isincubated with blocking buffer containing the nonionic detergent Tween20 and nonfat dry milk as a source of protein to later blockadventitious binding of specific antibodies to the nylon membrane. Theimmobilized proteins are then reacted with anti-phosphotyrosineantibodies and visualized after reaction with a secondary antibodyconjugated to horse radish peroxidase. Exposure to hydrogen peroxide inpresence of the chromogenic indicator diaminobenzidine produces visiblebands where secondary antibodies are bound, thereby enabling theirlocalization.

[0850] A variation of this assay can be performed with antibodiesdirected against phosphothreonine (for instance, those available fromSanta Cruz Biotechnology, catalog number sc-5267) or a host ofphosphorylated molecules. Sources of available phosphoprotein specificantibodies include, but are not limited to, Santa Cruz Biotechnology ofSanta Cruz, Calif., Calbiochem of San Diego, Calif. and ChemiconInternational, Inc. of Temecula, Calif.

[0851] The protein phosphorylation detection assays may be employedbefore and/or after treatment with an agent of interest to detectchanges in phosphorylation status of a polypeptide, or group ofpolypeptides. Moreover, detection of changes in phosphorylation statusof polypeptides of interest may be used to monitor efficacy of atherapeutic treatment or progression of a chronic disease.

[0852] (b) Immunoprecipitation

[0853] In one complementary embodiment, the relative levels ofphosphorylated and nonphosphorylated forms of any particular protein maybe measured. The levels of the phosphorylated forms are measured asdescribed above. Nonphosphorylated proteins are measured by firstimmunoprecipitating all forms of the protein of interest with a specificantibody directed toward that protein. The immune complexes are thenanalyzed by Western blotting as described. Comparison of the levels oftotal protein of interest to those of the phosphorylated forms providesome insight into the relative levels of each form of the polypeptide ofinterest.

[0854] (12) Assaying Gene Activation and Suppression

[0855] (a) Co-Transfection With Report Gene to Identify Transactivators

[0856] In one exemplary embodiment, interactions between regulatoryproteins and a DNA sequence of interest can be revealed throughco-transfection of two recombinant vectors. The first vector carries afull length cDNA for the regulatory factor driven by a promoter known tobe active in the transfected cells. The second recombinant vectorcarries a reporter gene driven by the DNA sequence of interest. Examplesof suitable reporter genes include chloramphenicol acetyltransferase(CAT), luciferase or β-galactosidase (Virts 2001²⁰⁴). Detection ofreporter gene expression by methods known in the art (see examplesbelow) indicates transactivation of the DNA sequence of interest by theregulatory factor.

[0857] Transfection of appropriate recombinant vectors can be mediatedeither with calcium phosphate (Chen 1988²⁰⁵) or DEAE-dextran (Lopata1984²⁰⁶). In one exemplary embodiment, exponentially growing cells areexposed to precipitated DNA. A DNA solution, prepared in 0.25M CaCl₂ isadded to an equal volume of HEPES buffered saline and incubated brieflyat room temperature. The mixture is then placed over cells and incubatedovernight to permit DNA adsorption and absorption into the cells. Thenext day the cells are washed and cultured in complete growth medium.

[0858] In a related exemplary embodiment, calcium chloride precipitationis replaced with DEAE-dextran as a carrier for the DNA to betransfected. Growth medium is made 2.5% with respect to fetal bovineserum (FBS) and 10 μM with respect to chloroquine. The medium isprewarmed, and DNA is added prior to addition of DEAE-dextran. Themixture is then added to exponentially growing cells, and incubated for4 hours to allow DNA adsorption. The transfection medium is replaced bya 10% solution of DMSO causing the DNA to enter the cells. The cells areincubated for 2-10 hours. The DMSO solution is then replaced by growthmedium, and the cells are incubated until assayed for exogenous geneexpression.

[0859] CAT

[0860] Detection of CAT gene expression is achieved by mixing lysates ofthe cells in which the reporter gene has been co-transfected along witha recombinant vector carrying the putative activating factor with¹⁴C-labeled chloramphenicol (Gorman 1982²⁰⁷). Acetylated andunacetylated forms of the compound, the latter resulting from enzymaticdegradation of the substrate by expressed CAT, are separated by thinlayer chromatography and visualized by autoradiography. Quantitation ofeach radiolabeled species is attained by densitometric analysis of theautoradiograph, or by direct phosphorimager analysis of thechromatograph.

[0861] Luciferase

[0862] Detection of expressed luciferase is achieved by exposure oftransfected cell lysates to the luciferase substrate luciferin inpresence of ATP, magnesium and molecular oxygen (Luo 2001²⁰⁸). Thepresence of luciferase results in transient release of light detected byluminometer.

[0863] β-Galactosidase

[0864] Detection of β-galactosidase gene expression is achieved bymixing cell lysates with a chromogenic substrate for the enzyme, such aso-nitrophenyl-β-D-galactopyranoside (ONPG), or a chemiluminescentsubstrate containing 1,2 dioxetane. Products of the catalyticdegradation of the chromogenic substrate are easily visualized, oralternatively, quantified by spectrophotometry, while the products ofthe chemiluminescent substrate are detected by luminometer. The latterassay is especially sensitive and can detect minute levels, or minutechanges in levels of β-galactosidase reporter gene expression.

[0865] These assays were applied to demonstrate binding of GABP to thepromoter regions of a number of genes including the retinoblastoma gene(Sowa 1997²⁰⁹), CD18 (Rosmarin 1998²¹⁰), cytochrome C oxidase Vb(Sucharov 1995²¹¹) and the prolactin gene (Ouyang 1996²¹²).

[0866] (b) Co-Transfection With Reporter Gene to Identify Trans-ActingRepressors

[0867] These assays can be applied to assess trans-acting factors whichpotentially repress rather than stimulate reporter gene expression. Inthis embodiment, putative repression factors are expressed from arecombinant vector in cells which carry a reporter gene driven by aconstitutively active promoter which may interact with the repressionfactor. The assays described above are applied to determine whetherexpression of the repression factor reduces reporter gene activity.

[0868] (13) Assaying Gene Expression Levels

[0869] (a) Northern Blot Analyses

[0870] In one exemplary embodiment, the relative expression levels of agene of interest are measured by Northern blot analysis (Ausubel 1999,Ibid). RNA is isolated from untreated cells and cells after treatmentwith an agent expected to modulate gene expression. The RNA is separatedby electrophoresis through a denaturing agarose gel, typicallyincorporating the denaturant formaldehyde, and transferred to a nylonmembrane. Immobilized RNA is hybridized to a radiolabeled DNA proberepresenting the gene of interest. Bound radiolabel is visualized byautoradiography. Levels of bound radiolabel can be quantified byscanning the resulting autoradiograph with a densitometer andintegrating the area under the traces. Alternatively, incorporatedradiolabel can be quantified by phosphorimager analysis of the blotitself.

[0871] (b) RT-PCR

[0872] In a related embodiment, RNA is isolated from similarly treatedcells. The RNA is then subjected to reverse transcription (RT) andamplification by the polymerase chain reaction (PCR) in the presence ofradiolabeled deoxynucleotides. The amplification products are resolvedby gel electrophoresis and visualized by autoradiography. Levels ofincorporated radiolabel can be quantified by scanning the resultingautoradiograph with a densitometer and integrating the area under thetraces. Alternatively, incorporated radiolabel can be quantified byphosphorimager analysis of the electrophoretic gel.

[0873] (14) Assaying Viral Replication

[0874] (a) Viral Titer

[0875] In one exemplary embodiment, viral replication is measured bytitration of infectious particles on cultured host cells. Virusreplication is permitted in host cells, with or without chemicaltreatment, or with or without co-expression of a regulatory gene, for ameasured period of time. The cells are lysed by exposure to a hypotonicsolution, and the lysates are subjected to a series of dilutions inisotonic buffer. Several concentrations of cell lysate are separatelyplated onto cultured host cells. The culture cells are incubated untilthe cytopathic effects (CPE) are evident. The cultured cells are thenfixed and stained with a contrast enhancing dye, such as crystal violet,to facilitate identification of viral plaques. Several culture platesare counted, and the number of plaques multiplied by the appropriatedilution factor, representing the dilution from the original celllysate. The result reveals the viral titer of the original cell lysate.

[0876] (b) In situ PCR

[0877] In a related exemplary embodiment, a latent, low copy numbervirus can be detected with the polymerase chain reaction in situ(Staskus 1994²¹³) Cells grown either in suspension culture or on a solidsubstrate are fixed and permeabilized. PCR reaction components,including synthetic primers complementary to the gene of interest, Taqpolymerase, deoxyribonucleotides, are then added to the cells andsubjected to thermal cycling typical of PCR. The amplification products,retained in each cell, are detected by in situ hybridization withappropriately labeled DNA probes. An exemplary detection method involveshybridization with radiolabeled probes followed by autoradiography.Similarly, hybridization probes may be nonradioactively labeled byincluding digoxygenin-11-dUTP into the PCR reaction. Incorporated labelis detected either enzymatically or chemically.

[0878] (15) Assaying Cell Morphology and Function

[0879] (a) Light Microscopy

[0880] In one exemplary embodiment, the morphology of cells isascertained by microscopic examination. Living and dead cells aredistinguished by treating cells with the stain trypan blue (Schuurhuis2001²¹⁴). Living cells, with intact cellular membranes, exclude trypanblue while dead cells, with leaky, or perforated outer membranes, permittrypan blue to enter the cytoplasm. Following treatment, examination byphase contrast microscopy reveals the proportion of dead vs. livingcells. Similarly, cellular morphology can be ascertained by examinationwith phase contrast microscopy, with or without prior staining, with,for example, crystal violet, to enhance contrast. Such examinationreveals morphologies common to known cell types, and concomitantlyreveals irregularities present in the cell population under examination.

[0881] (b) Functional Assessment by Immunocytochemistry

[0882] In a related exemplary embodiment, the functional status of agiven cell population may be determined by treatment with specificantibodies. Cells are dehydrated and fixed with a series of methanolwashes using increasing concentrations of methanol. Once fixed, thecells are exposed to cell-type specific antibodies. Examples of suitableantibodies include, but are not limited to, anti-filaggrin for epidermalcells, anti-CD4 for T cells, thymocytes and monocytes, andanti-macrosialin for macrophages. After incubation withdifferentiation-specific marker antibodies, fluorescently labeledsecondary antibodies specific for the first antibody are added. Boundsecondary antibodies are visualized by illumination with light ofappropriate wavelength to excite the bound fluorochrome followed bymicroscopic examination. The use of different antibodies, eachconjugated to a different fluorochrome, permits the identification ofmultiple differentiation-specific antigens simultaneously in the samepopulation of cells.

[0883] (16) Assaying Cellular Oxidation Stress

[0884] (a) Cellular Indicators

[0885] In one exemplary embodiment, oxidation stress within a populationof cells can be measured by assaying the activity levels of certainindicators such as lipid hydroperoxides (Weyers 2001²¹⁵). Cell lysatesare prepared and mixed with the substrate 1-napthyldiphenylphosphiine(NDPP). Any resulting oxidized form of the substrate, ONDPP, can bequantified by high performance liquid chromatography (HPLC). ONDPPconcentration provides an indirect measure of the oxidation capacity ofthe cell lysate.

[0886] (b) H2DCFDA as Indicator

[0887] In another exemplary embodiment, the production of cellularreactive oxygen species can be detected by mixing cell lysates with2′,7′-dichlorodihydrofleuoescein diacetate (H2DCFDA) (Brubacher2001²¹⁶). In the presence of cellular esterases, H2DCFDA is deacetlyatedto produce 2′,7′-dichlorodihydrofleuoescein (H2DCF), anoxidant-sensitive indicator. Increased cellular oxidation excites thefluorogenic indicator. Increased sensitivity can be attained by usingH2DCF directly, but caution must be exercised by one skilled in the artto ensure that none of the experimental buffers contain contaminants,such as metals, which may lead to spontaneous fluorescence.

d) Optimization Protocols

[0888] Once a single constructive or disruptive agent (polynucleotide,polypeptide, small molecule, etc.) is identified in the manner describedabove, variant agents can be formulated that improve upon the originalagent.

[0889] The expression “variant agents . . . that improve upon theoriginal agent” is understood to include, but not be limited to, agentsthat increase therapeutic efficacy, increase prophylactic potential,increase, or decrease stability in vivo or in storage, or increase thenumber, or variety of post-translational modifications in vivo,including, but not limited to, phosphorylation, acetylation andglycosylation, relative to the original agent.

[0890] Variant agents are not limited to those produced in thelaboratory. They may include naturally occurring variants. For example,variants with increased stability, due to alterations in ubiquitinationor modifications of other target sites conferring resistance toproteolytic degradation.

e) Treatment Protocols

[0891] (1) Introduction

[0892] According to the present invention, a polypeptide has aconstructive effect if it attenuates microcompetition with a foreignpolynucleotide or attenuates at least one effect of microcompetitionwith a foreign polynucleotide, or one effect of another foreignpolynucleotide-type disruption. For example, a constructive polypeptidecan reduce copy number of the foreign polynucleotide, stimulateexpression of a GABP regulated gene, increase bioactivity of a GABPregulated protein, through, for instance, GABP phosphorylation and/orincrease bioavailability of a GABP regulated protein, through, forinstance, a reduction in copy number of microcompeting foreignpolynucleotides which bind GABP. A constructive polypeptide can also,for example, inhibit expression of a microcompetition suppressed gene,such as, tissue factor, androgen receptor, and/or inhibit replication ofa p300/cbp virus (see more examples below).

[0893] Agents of the present invention are designed to address andameliorate symptoms of chronic diseases, specifically, diseasesresulting from microcompetition between a foreign polynucleotide andcellular genes. For instance, introduction of an oligonucleotide agentinto a cell may disrupt this microcompetition and restore normalregulation and expression of a microcompeted gene. Agents directedagainst a foreign polynucleotide may reduce binding or cellulartranscription factors to the foreign polynucleotide by, for instance,reducing the copy number of the foreign polynucleotide, or its affinityto the transcription factor, resulting in increased microavailability ofthe factors towards normal levels. Alternatively, binding of thetranscription factors to cellular genes can be stimulated. In yetanother exemplary embodiment, insufficient, or excessive expression of acellular gene in a subject can be modified by administration of nucleicacids or polypeptides to the subject that return the concentration of acellular polypeptide of interest towards normal levels.

[0894] The following section describes standard protocols fordetermining effective dose, and for agent formulation for use.Additional standard protocols and background information are availablein books, such as In vitro Toxicity Testing Protocols (Methods inMolecular Medicine, 43), edited by Sheila O'Hare and C K Atterwill,Humana Press, 1995; Current Protocols in Pharmacology, edited by: S JEnna, Michael Williams, John W Ferkany, Terry Kenakin, Roger D Porsolt,James P Sullivan; Current Protocols in Toxicology, edited by: MahinMaines (Editor-in-Chief), Lucio G Costa, Donald J Reed, Shigeru Sassa, IGlenn Sipes; Remington: The Science and Practice of Pharmacy, edited byAlfonso R Gennaro, 20^(th) edition, Lippincott, Williams & WilkinsPublishers, 2000, Pharmaceutical Dosage Forms and Drug Delivery Systems,by Howard C Ansel, Loyd V Allen, Nicholas G Popovich, 7^(th) edition,Lippincott Williams & Wilkins Publishers, 1999, PharmaceuticalCalculations, by Mitchell J Stoklosa, Howard C Ansel, 10^(th) edition,Lippincott, Williams & Wilkins Publishers, 1996, AppliedBiopharmaceutics and Pharmacokinetics, by Leon Shargel, Andrew B C Yu,4^(th) edition, McGraw-Hill Professional Publishing, 1999, Oral DrugAbsorption: Prediction and Assessment (Drugs and the PharmaceuticalSciences, Vol 106), edited by Jennifer B Dressman, Hans Lennernas,Marcel Dekker, 2000, Goodman & Gilman's The Pharmacological Basis ofTherapeutics, edited by Joel G Hardman, Lee E Limbird, 10^(th) edition,McGraw-Hill Professional Publishing, 2001. See also above referenced.

[0895] (2) Effective Dose

[0896] Compounds can be administered to a subject, at a therapeuticallyeffective dose, to treat, ameliorate, or prevent a chronic disease.Careful monitoring of patient status, using either systemic means,standard clinical laboratory assays or assays specifically designed tomonitor the bioactivity of a foreign polynucleotide, is necessary toestablish the therapeutic dose and monitor its effectiveness.

[0897] Prior to patient administration, techniques standard in the artare used with any agent described herein to determine the LD₅₀ and ED₅₀(lethal dose which kills one half the treated population, and effectivedose in one half the population, respectively) either in cultured cellsor laboratory animals. The ratio LD₅₀/ED₅₀ represents the therapeuticindex which indicates the ratio between toxic and therapeutic effects.Compounds with a relatively large index are preferred. These values arealso used to determine the initial therapeutic dose. While unwanted sideeffects are sometimes unavoidable, they may be minimized by delivery ofthe therapeutic agent directly to target cells or tissues, therebyavoiding systemic exposure.

[0898] Those skilled in the art recognize that animal or cell culturemodels are imperfect predictors of the efficacy of any treatment inhumans. Factors affecting efficacy include route of administration,achievable serum concentration and formulation of the therapeutic agent(i.e. in pill or injectable forms, administered orally orintramuscularly, with accompanying carrier, formulation of an agentadducted with a specific antibody and injected directly into the targettissue, etc.). Regardless of the method of delivery or formulation ofthe therapeutic agent, it is important to monitor plasma levels using asuitable technique, such as atomic absorption spectroscopy, enzymelinked immunosorbant assay (ELISA), or high performance liquidchromatography (HPLC) among others.

[0899] (3) Formulation for Use

[0900] Those skilled in the art recognize a host of standardformulations for the agents described in this invention. Any suitableformulation may be prepared for delivery of the agent by injection,inhalation, transdermal diffusion or insufflation. In every case, theformulation must be appropriate for the means and route ofadministration.

[0901] Oligonucleotide agents, e.g. antisense oligonucleotides orrecombinant expression vectors, may be formulated for localized orsystemic administration. Systemic administration may be achieved byinjection in a physiologically isotonic buffer including Ringer's orHank's solution, among others. Alternatively, the agent may be givenorally by delivery in a tablet, capsule or liquid syrup. Those skilledin the art recognize pharmaceutical binding agents and carriers whichprotect the agent from degradation in the digestive system andfacilitate uptake. Similarly, coatings for the tablet or capsule may beused to ease ingestion thereby encouraging patient compliance. Ifdelivered in liquid suspension, additives may be included which keep theagent suspended, such as sorbitol syrup and the emulsifying agentlecithin, among others, lipophilic additives may be included, such asoily esters, or preservatives may be used to increase shelf life of theagent. Patient compliance may be further enhanced by the addition offlavors, coloring agents or sweeteners. In a related embodiment theagent may be provided in lyophilized form for reconstitution by thepatient or his or her caregiver.

[0902] The agents described herein may also be delivered via buccalabsorption in lozenge form or by inhalation via nasal aerosol. In thelatter mode of administration any of several propellants, including, butnot limited to, trichlorofluoromethane and carbon dioxide, or deliverymethods, including but not limited to a nebulser, can be employed.Similarly, compounds may be included in the formulation which facilitatetransepithelial uptake of the agent. These include, among others, bilesalts and detergents. Alternatively, the agents of this invention may beformulated for delivery by rectal suppository or retention enema. Thoseskilled in the art recognize suitable methods for delivery of controlleddoses.

[0903] In related embodiments, the agents may be formulated for depotadministration, such as by implantation, via regulated pumps, eitherimplanted or worn extracorporally or by intramuscular injection. Inthese instances the agent may be formulated with hydrophobic materials,such as an emulsification in a pharmaceutically permissible oil, boundto ion exchange resins or as a sparingly soluble salt.

[0904] In every case, therapeutic agents destined for administrationoutside of a clinical setting may be packaged in any suitable way thatassures patient compliance with regard to dose and frequency ofadministration.

[0905] Administration of the agents included in this invention in aclinical situation may be achieved by a number of means includinginjection. This method of systemic administration may achieve cell-typespecific targeting by using a nucleic acid agent, described herein,modified by addition of a polypeptide which binds to receptors on thetarget cell. Additional specificity may be derived from the use ofrecombinant expression vectors which carry cell- or tissue-type specificpromoters or other regulatory elements.

[0906] In contrast to systemic injection more specific delivery may beachieved by means of a catheter, by stereotactic injection, byelectorporation or by transdermal electrophoresis. Many suitabledelivery techniques are well known in the art.

[0907] In an alternative embodiment the therapeutic agent may beadministered by infection with a recombinant virus carrying the agent.Similarly cells may be engineered ex vivo which express the agent. Thosecells may themselves become the pharmaceutical agent for implantationinto the site of interest in the patient.

f) Diagnosis Protocols

[0908] Diagnosis may be achieved by a number of methods, well known inthe art, using as reagents sequences of a foreign polynucleotide,disrupted gene or polypeptide, or a gene or polypeptide in a disruptiveor disrupted pathway, or antibodies directed against suchpolynucleotides or polypeptides. Those reagents may be used to detectand quantify the copy number, level of expression or persistence ofexpression products of a foreign polynucleotide, disrupted gene or genesusceptible to microcompetition with a foreign polynucleotide.

[0909] Diagnostic methods may employ any suitable technique well knownin the art. These include, but are not limited to, commerciallyavailable diagnostic kits which are specific for one or more foreignpolynucleotides, a specific disrupted gene, a disrupted polypeptide, agene or polypeptide in a disruptive or disrupted pathway, or an antibodyagainst such polynucleotides or polypeptides. Well known advantages ofcommercial kits include convenience and reproducibility due tomanufacturing standardization, quality control and validationprocedures.

[0910] (1) Detection and Quantification of Polynucleotides

[0911] In one exemplary embodiment, nucleic acids, DNA or RNA, areisolated from a cell or tissue of interest using procedures well knownin the art. Once isolated, the presence of a foreign polynucleotide maybe ascertained by any of a number of procedures including, but notlimited to, Southern blot hybridization, dot blotting and the PCR, amongothers.

[0912] Mutations in those polynucleotides may be detected by singlestrand conformation analysis, allele specific oligonucleotidehybridization and related and complementary techniques. Alternativelynucleic acid hybridization with appropriately labeled probes may beperformed in situ on isolated cells or tissues removed from the patient.Suitable techniques are described, for example, Sambrook 2001 (ibid),incorporated herein in its entirety by reference. Control cells andtissues are compared in parallel to validate any positive findings inclinical samples.

[0913] If the nucleic acid molecules specific to foreign polynucleotidesor disrupted genes, or genes in disrupted or disruptive pathways are inlow concentration, preferred diagnostic methods employ some means ofamplification. Examples of suitable procedures include the PCR, ligasechain reaction, or any of a number of other suitable methods well knownin the art.

[0914] In one exemplary embodiment of a diagnostic technique employingnucleic acid hybridization, RNA from the cell of interest is isolatedand converted to cDNA (using the enzyme reverse transcriptase of avianor murine origin). Once cDNA is prepared, it is amplified by the PCR, ora similar method, using a sequence specific oligonucleotide primer of20-30 nucleotides in length. Incorporation of radiolabeled nucleotidesduring amplification facilitates detection following electrophoresisthrough native polyacrylamide gels by autoradiography or phosphorimageranalysis. If sufficient amplification products are attained, they may bevisualized by staining of the electrophoretic gel by ethidium bromide ora similar compound well known in the art.

[0915] (2) Detection and Quantification of Polypeptides

[0916] Antibodies directed against foreign polypeptides, disruptedpolypeptides, or polypeptides in disrupted or disruptive pathways, mayalso be used for the diagnosis of chronic disease. Diagnostic protocolsmay be employed to detect variations in the expression levels ofpolypeptides or RNA transcripts. Similarly, they may be used to detectstructural variation including nucleic acid mutations and changes in thesequence of encoded polypeptides. The latter may be detected by changesin electrophoretic mobility, indicative of altered charge, or by changesin immunoreactivity, indicative of alterations in antigenicdeterminants.

[0917] For diagnositic purposes, protein may be isolated from the cellsor tissues of interest using any of many techniques well known in theart. Exemplary protocols are described in Molecular Cloning: ALaboratory Manual, 3rd Ed (Third Edition) By Joe Sambrook, PeterMacCallum and David Russell (Cold Spring Harbor Laboratory Press 2001),incorporated herein by reference in its entirety.

[0918] In a preferred embodiment, detection of a foreign polypeptidemolecule, or a cellular disrupted polypeptide molecule, or a polypeptidein a disruptive or disrupted pathway is achieved with immunologicalmethods, including immunoaffinity chromatography, radial immunoassays,radioimmunoassay, enzyme linked immunsorbant assay, etc. Thesetechniques, quantitative and qualitative, all well known in the art,exploit the interaction between specific antibodies and antigenicdeterminants on the target molecule. In each assay, polyclonal ormonoclonal antibodies, or fragments thereof, may be used as appropriate.

[0919] Immunological assays may be employed to analyze histologicalpreparations. In a preferred embodiment, tissue or cells of interest aretreated with a fluorescently labeled specific antibody or an unlabeledantibody followed by reaction with a secondary fluorescently labeledantibody. Following incubation for sufficient time and under appropriateconditions for antibody-antigen interaction, the label may be visualizedmicroscopically, in the case of either tissues or cells, or by flowcytometry, in the case of individual cells. These techniques areparticularly suitable for antigens expressed on the cell surface. Ifthey are not on the cell surface, the cells or tissue to be analyzedmust be treated to become permeable to the diagnostic antibodies. Inaddition to the detection of antigens on the material studied, thedistribution of that antibody will become evident upon microscopicexamination. All immunological assays involve the incubation of abiological sample, cells or tissue, with an appropriately specificantibody or antibodies. These and other suitable diagnostic methods arefamiliar to those skilled in the art.

[0920] In an alternative embodiment, immunological techniques may beemployed which involve either immobilized antibodies or immobilizing thecells to be analyzed on, for example, synthetic beads or the surface ofa plastic dish, typically a microtiter plate (see above).

[0921] Immobilization of antibodies or cells to be analyzed is achievedthrough the use of any of several substrates well known in the artincluding, but not limited to, glass, dextran, nylon, cellulose, andpolypropylene, among others. The actual shape or configuration of thesubstrate may vary to suite the desired assay. For example, polystyrenemay be formed into tissue culture or microtitre plates, dextran may beformed into beads suitable for column chromatography, or polyacrylamidemay be coated onto the inner surface of a glass test tube or bottle.These and related carriers and configurations are well known and can betested for utility by those skilled in the art.

[0922] Detection of bound antibodies is achieved by labeling, eitherdirectly or indirectly, through the use of a secondary antibody specificfor the first. The label may be either a chromophore which responds toexcitation by a specific wavelength of light, thereby producingfluorescence or it may be an enzyme which reacts with a chromogenicsubstrate to produce detectable reaction products. Common florescentlabels include fluorescineisothiocyanate (FITC), rhodamine andtrans-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (BSB),among others. Enzymes commonly conjugated with antibodies include, butare not limited to, alkaline phosphatase, horse radish peroxidase andβ-galactosidase. Other alternatives are available and well known in theart.

[0923] In a related embodiment, the antibody is labeled with afluorescent metal, for example ¹⁵²Eu, which can be attached directly tothe primary or secondary antibody in an immunoassay. Alternatively, theantibody may be labeled with a chemiluminescent compound, such asluminol, isoluminol or imidazole or a bioluminiscent compount, such asluciferin or aequorin. Subsequent reaction with the appropriatesubstrate for the labeling compound produces light which is detectablevisually or by fluorimetry.

[0924] (3) Imaging of Diseased Tissues

[0925] Under suitable circumstances, foreign polypeptides, polypeptidesexpressed from disrupted genes, or from genes in a disruptive ordisrupted pathway, may be detected on the surface of affected cells ortissues. In these instances the level and pattern of expression may bevisualized and used to both diagnose disease and to guide and gaugetherapy. For example, in atherosclerosis, such disrupted polypeptidesmay include, but are not limited to CD18 or tissue factor (see moredetails in examples below).

[0926] Under these circumstances, antibodies, monoclonal or polyclonal,which specifically interact with proteins expressed on the cell surfacemay be used for the diagnosis of chronic disease and for monitoringtreatment efficacy. In this embodiment, an appropriate antibody orantibody fragment is labeled with a radioactive, fluorescent, or othersuitable tag prior to reaction with the biomaterial to be assayed.Conditions for reaction and visualization are well known in the art andpermit analyses to be carried out in vitro as well as in situ. In apreferred embodiment, antibody fragments are used for in situ or invitro assays because their smaller size leads to more rapid accumulationin the tissue of interest and more rapid clearing from that tissuefollowing the assay. A number of suitable and appropriate labels may beused for the assays in this invention that are well known in the art.

g) Clinical Trials

[0927] Another aspect of current invention involves monitoring theeffect of a compound on a treated subject in a clinical trial. In such atrial, the copy number of a foreign polynucleotide, its affinity tocellular transcription factors, the expression or bioactivity of adisrupted gene or polypeptide, or expression or bioactivity of a gene orpolypeptide in a disrupted or disruptive pathway, may be used as anindicator of the compound effect on a disease state.

[0928] For example, to study the effect of a test compound in a clinicaltrial, blood may be collected from a subject before, and at differenttimes following treatment with such a compound. The copy number of aforeign polynucleotide may be assayed in monocytes as described above,or the levels of expression of a disrupted gene, such as tissue factor,may be assayed by, for instance, Northern blot analysis, or RT-PCR, asdescribed in this application, or by measuring the concentration of theprotein by one of the methods described above. In this way, the copynumber, or expression profile of a gene of interest or its mRNA, mayserve a surrogate or direct biomarker of treatment efficacy.Accordingly, the response may be determined prior to, and at varioustimes following compound administration. The effects of any therapeuticagent of this invention may be similarly studied if, prior to the study,a suitable surrogate or direct biomarker of efficacy, which is readilyassayable, was identified.

B. Examples

[0929] The current view holds that, in vivo, viral proteins are the solemediators of viral effects on the host cell. Such proteins include, forexample, the papilomavirus type 16 E6 and E7 oncoproteins, SV40 large Tantigen, Epstein-Barr virus BRLF1 protein, and adenovirus E1A. Thepossibility that presence of viral DNA in the host cell can directlyimpact cell function, independent of viral protein, is typicallyignored. The viral “protein-dependent” view is so ingrained in currentresearch that in many cases, when a “protein-independent” effect oncellular gene expression, or other cell functions, presents itself inthe laboratory, the effect is ignored. As a result, the significance ofsuch effect, and specifically, its relation to disease is overlooked.Note that the effect of viral DNA on the cellular genome in cases ofviral DNA integration which may result in mutations, deletions ormethylation of host cell DNA, cannot be considered “protein-independent”since it is mediated by viral proteins, such as, HIV-1 IN protein, orretrovirus integrase.

[0930] The following examples illustrate the invention. More examplescan be found in patent application PCT/US01/05314, incorporated hereinin its entirety by reference.

1. Foreign Polynucleotides and Aberrant Transcription a) Introduction

[0931] Microcompetition between a foreign polynucleotide and a cellulargene for a limiting cellular transcription complex result in aberranttranscription of the cellular gene. If the limiting complex stimulatesthe gene transcription, microcompetition with the foreign polynucleotidereduces transcription. If the limiting complex suppresses the genetranscription, microcompetition with the the foreign polynucleotideincreases transcription. Aberrant transcription can result in abberantgene expression, abnormal gene product activity, and irregular cellfunction. Consider the following observations.

b) Examples

[0932] (1) Scholer 1984

[0933] The plasmid pSV2CAT expresses the chloramphenicolacethyltransferase (CAT) gene under the control of the SV40promoter/enhancer. A study (Scholer 1984²¹⁷) first transfected anincreasing amount of pSV2CAT in CV-1 cells. CAT activity reached aplateau at 0.3 pmol pSV2CAT DNA per dish. Based on this observation, thestudy concluded that CV-1 cell contain a limited concentration ofcellular factor needed for pSV2CAT transcription. Next, the studycotransfected a constant concentration of pSV2CAT with increasingconcentrations of pSV2neo, a plasmid identical to pSV2CAT, except thereporter gene is neomycin-phosphotransferase (neo). The addition ofpSV2neo resulted in a linear decrease of the CAT signal (Scholer 1984,ibid, FIG. 2B). Next, the study cotransfected pSV2CAT with a plasmidthat included all SV40 early control elements except for the 72-bpenhancer. No competition was observed. A point mutation in the 72-bpenhancer, which abolished the enhancer functional activity, alsoeliminated competition. Based on these observations, Scholer, et al.,(1984, ibid) concluded that “taken together, our data indicate that alimited amount of the cellular factors required for the function of theSV40 72-bp repeats is present in CV-1 cells. Increasing the number offunctional SV40 enhancer elements successfully competes for thesefactors, whereas other elements necessary for stable transcription didnot show such an effect.” The study also observed competition betweenpSV2CAT and pSrM2Δ, a plasmid which harbors the Moloney murine sarcomavirus (MSV) enhancer (Scholer 1984, ibid, FIG. 5A and B). Note, thatexcept the enhancers, the transcriptional control elements in pSV2CATand pSrM2Δ are same. Based on these observations, Scholer, et al.,(1984, ibid) concluded that “one class of (a limiting) cellularfactor(s) is required for the activity of different enhancers.Furthermore, BK (BK virus) and RSV (Rous sarcoma virus) enhancers alsointeract with the same class of molecule(s).”

[0934] (2) Mercola 1985

[0935] The plasmid pSV2CAT expresses the chloramphenicolacethyltransferase (CAT) gene under the control of the SV40promoter/enhancer. The pX1.0 plasmid contains the murine immunoglobulinheavy-chain (Ig H) enhancer. The pSV2neo expresses the neo gene underthe control of the SV40 promoter/enhancer. The pA10neo and pSV2neo areidentical except that pA10neo lacks most of the SV40 enhancer.

[0936] A study (Mercola 1985²¹⁸) cotransfected a constant amount ofpSV2CAT into murine plasmacytoma P3X63-Ag8 cells, as test plasmid, withincreasing amounts of pX1.0 as competitor plasmid. A plasmid lackingboth reporter gene and enhancer sequences was added to produce equimolaramounts of plasmid DNA in the transfected cells. FIG. 1 illustrates theobserved relative CAT activity as a function of the relativeconcentration of the competitor plasmid (Mercola 1985, ibid, FIG. 4A).

[0937] An increase in concentration of the contransfected murineimmunoglobulin heavy-chain (H) enhancer decreased expression from theplasmid carrying the SV40 viral enhancer. Microcompetition between viraland cellular heavy-chain enhancers decreased expression of the geneunder control of the viral enhancer. Based on these observations,Mercola, et al., (1985, ibid) concluded that in the plasmacytoma cellsthe heavy chain enhancer competes for a trans-acting factor required forthe SV40 enhancer function.

[0938] In another experiment, the study cotransfected a constant amountof pSV2CAT, as test plasmid, with increasing amount of pSV2neo, ascompetitior plasmid, in either Ltk- or ML fibroblast cells. To isolatethe effect of the viral enhancer, the study also cotransfected aconstant amount of the test plasmid pSV2CAT with increasing amount ofthe enhancerless pA10neo plasmid. FIG. 2 illustrates the observedrelative CAT activity as a function of the relative concentration of thecompetitor plasmid (Mercola 1985, ibid, FIG. 4B).

[0939] An increase in concentration of the contransfected SV40 viralenhancer decreased expression from the plasmid also carrying the SV40enhancer. An increase in concentration of a plasmid lacking the enhancerdid not affect the test plasmid reporter gene activity.

[0940] Overall, the study concluded that “in vivo competitionexperiments revealed the presence of a limited concentration ofmolecules that bind to the heavy-chain enhancer and are required for itsactivity. In the plasmacytoma cell, transcription dependent on the SV40enhancer was also prevented with the heavy-chain enhancer as competitor,indicating that at least one common factor is utilized by theheavy-chain and SV40 enhancers.”

[0941] (3) Scholer 1986

[0942] Another study (Scholer 1986²¹⁹) cotransfected CV-1 monkey kidneycells with a constant amount of a plasmid containing the hMT-II_(A)promoter (−286 nt relative to the start of transcription to +75 nt)fused to the bacterial gene encoding chloramphenicol acetyltransferase(hMT-IIA-CAT) along with increasing concentrations of a plasmidcontaining the viral SV40 early promoter and enhancer fused to thebacterial gene conferring neomycin resistance (pSV2neo). FIG. 3 presentsthe observed relative CAT activity (expressed as the ratio between CATactivity in the presence of pSV2neo and CAT activity in the absence ofpSV2neo) as a function of the molar ratio of pSV2Neo to hMT-IIA-CAT.

[0943] The figure illustrates the effect of competition between the twoplasmids on the relative CAT activity. A 2.4-fold molar excess of theplasmid containing the viral enhancer reduced CAT activity by 90%. Noocompetition was observed with the viral plasmid after deletion of theSV40 enhancer suggesting that elements in the viral enhancer areresponsible for the observed reduction in reporter gene expression.

[0944] (4) Cherington 1988

[0945] pZIP-neo expresses the neomycin-resistant gene under control ofthe Moloney murine leukemia virus long terminal repeat (LTR) (Cepko1984²²⁰).

[0946] Another study inserted the wild-type early region of SV40 intothe “empty” pZIP-Neo plasmid and labled the new plasmid, which expressedthe SV40 large T antigen, “wild-type” (WT). The study transfected3T3-F442A preadipocytes with either WT or pZIP-neo. Accumulation oftriglyceride, assayed by oil red staining, was used as marker ofdifferentiation. Seven days postconfluence, the number of staining ofcells was recorded. Consider the following figure (Cherington 1988²²¹,FIG. 4 A, B and C). Darker staining indicates increased differentiation.(A) marks untreated F442A cells, (B) marks cells transfected withpZIP-neo, (C) marks cells transfected with WT. Consider FIG. 4.

[0947] Transfection with WT, the vector expressing the SV40 large Tantigen, reduced differentiation. Compare triglyceride staining in (C)and (A). Transfection with the “empty” vector, although less so thantransfection with the WT vector, also reduced differentiation. Comparetriglyceride staining in (B) relative to (A) and (C). The resultsdemotstrate the effect of microcomopetition on cell differentiation.

[0948] (5) Adam 1996

[0949] Another study (Adam 1996²²²) cotransfected JEG-3 humanchoriocarcinoma cells with a constant concentration of a plasmidcarrying CAT reporter gene under the control of the platelet derivedgrowth factor-B (PDGF-B) promoter/enhancer (PDGF-B-CAT), and increasingconcentrations of a second plasmid containing either the humancytomegalovirus promoter/enhancer fused to β-galactosidase (CMV-βgal),or the SV40 early promoter and enhancer elements fused to βgal(SV40-βgal). FIG. 5 present the observed relative CAT activity as afunction of the molar ratio between the plasmids carrying βgal and CAT(based on Adam 1996, ibid, FIG. 1).

[0950] The results demonstrate the negative, concentration-dependenteffect of microcompetition between the CMV promoter/enhancer, or SV40promoter/enhancer, and the PDGF-B promoter.

[0951] (6) Higgins 1996

[0952] HSV-neo is a plasmid that expresses the neomycin-resistance geneunder control of the murine Harvey sarcoma virus long terminal repeat(LTR) (Armelin 1984²²³). pZIP-neo expresses the neomycin-resistant geneunder control of the Moloney murine leukemia virus long terminal repeat(LTR) (Cepko 1984, ibid).

[0953] A study (Higgins 1996²²⁴) transfected murine 3T3-L1 preadipocyteswith PVU0, a vector carrying an intact early region of the SV40 genomeexpressing the SV40 large tumor antigen and the SV40 small tumorantigen. The cells were also transfected with HSV-neo and pZIP-neo as“empty” controls. Following transfection, the study cultured the cellsunder differentiation inducing conditions, and measured glycerophosphatedehydrogenase (GPD) activity as marker of differentiation. The resultsare presented in the following table (Higgins 1996²²⁵, Table 1, firstfour lines). GPD activity Vector Cell line (U/mg of protein) None L1 2,063 1,599 HSV-neo L1-HNeo  1,519 1,133 ZIP-neo L1-ZNeo  1,155 1,123PVU0 L1-PVU0 47,25

[0954] Transfection of PVU1 and expression of the large and small Tantigens resulted in a statistically significant decrease in GPDactivity. Transfection of the “empty” vectors, HSV-neo and ZIP-neo,although less effective than PVU0, also reduced GPD activity. In at-test, assuming unequal variances, the p-value for the differencebetween the HSV-neo vector and no vector is 0.118, and the p-value forthe difference between ZIP-neo and no vector is 0.103. Given that thesample includes only two observations, a p-value around 10% for vectorscarrying two different LTRs indicates a trend. The observationsdemonstrate the effect of microcompetition with HSV-neo and Zip-Neo, the“empty vectors,” on cell differentiation.

[0955] (7) Gordeladze 1997

[0956] The effect of microcompetition on hormone senstivie lipase (HSL)transcription can be demonstrated by combining observations from twostudies. Swiss mouse embryo 3T3-L1 fibroblasts can be induced todifferentiate into adipocyte-like cells. Undifferentiated cells containvery low level of HSL activity, while differentiated adipocyte-likecells show much higher activity (a 19-fold increase relative toundifferentiated cells) (Kawamura 1981²²⁶). 3T3-L1 preadipocytes wereinduced to differentiate by incubation with insulin (10 μg/ml),dexamethasone (10 nM), or iBuMeXan (0.5 mM) for 8 consecutive daysfollowing cell confluency. HSL mRNA was measured in undifferentiatedconfluent controls and differentiated 3T3-L1 cells transfected with thepZipNeo vector. Although differentiated 3T3-L1 cells usually showsignificant HSL activity, the 3T3-L1 differentiated cells transfectedwith pZipNeo showed decreased HSL mRNA (Gordeladze 1997²²⁷, FIG. 11.Compare pZipNeo and Wtype columns in FIG. 6).

[0957] pZipNeo carries the Moloney murine leukemia virus LTR whichmicrocompeted with 25 HSL promoter. The results demostrate the effect ofmicrocompetition with the viral LTR on HSL transcription.

[0958] (8) Awazu 1998

[0959] A study (Awazu 1998²²⁸) transfected HuH-7 human hepatoma cellswith pBARB, a plasmid in which the β-actin promoter regulates theexpression of the Rb gene, and the simian virus (SV40) promoterregulates the expression of the neomycin-resistance (neo) gene. Thestudy also transfected the cells with the pSV40-neo plasmid, which onlyincludes the SV40 promoter and neo gene. Since pSV40-neo does notinclude the β-actin promoter and Rb gene, the study considered thepSV40-neo plasmid as “empty” and used it as control. The cell wereincubated in IS-RPMI, with or without 5% FBS, and the number of viablecells were counted at the indicated times. FIG. 7 summarizes the results(Awazu 1998, ibid, FIG. 2A). The SD is about the size of the triangularsymbols.

[0960] The result demonstrate the effect of microcompetition withpSV40-neo, the “empty vector” on cell proliferation.

[0961] (9) Hofman 2000

[0962] The pSG5 plasmid includes the early SV40 promoter to facilitatein vivo expression, and the T7 bacteriophage promoter to facilitate invitro transcription of cloned inserts. Both the pcDNA1.1 and pIRESneoplasmids includes the human cytomegalovirus (CMV) immediate early (IE)promoter and enhancer.

[0963] Another study (Hofman 2000²²⁹) constructed a series of pSG5 basedvectors by cloning certain sequences into the EcoRI restriction site(“insert plasmid,” see list in table below). The inserts varied inlength measured in base pair (bp). The study cotransfected each insertplasmid (650 ng) with pSG5-luc (20 ng), as test plasmid, in COS-7 cells.The test plasmid pSG5-luc was also cotransfected with the pGEM-7Zf(+)plasmid, or with herring sperm DNA. Luciferase (luc) activities weremeasured. Luc activity in presence of the empty pSG5 vector wasarbitrarily set to 1. The following table presents the observed relativeluc activity in every experiment (Hofman 2000, ibid, FIG. 3a). Lucactivity Size of insert from pSG5-luc Plasmid (bp) (fold increase)pGEM7zf+ 72 herring 71 pSG5-NuRIP183 4,776 47 pSG5-TIF2 4,395 40pSG5-NuRIP183D1 4,326 36 pSG5-NuRIP183D2 3,723 33 pSG5-NuRIP183D3 3,21930 pSG5-NuRIP183D4 2,684 28 pSG5-NuRIP183D5 2,400 25 pSG5-NuRIP183D61,889 22 pSG5-ARA70 1,800 20 pSG5-TIF2.5   738  7 pSG5-DBI   259  3 pSG5   0  1

[0964] Based on these observations Hofman, et al., (2000, ibid)concluded that “Remarkably, the measured luciferase activity tended tobe inversely related to the length of the insert in the cotransfectedpSG5-constructs.” Moreover, “We can conclude from these data that theSV40 promoter driven expression of nuclear receptor or of luciferase inCOS-7 cells is inhibited to various degrees by cotransfection, withmaximal inhibition in the presence of the empty expression vector andminimal inhibition in the presence of pSG5 constructs containing largeinserts.” First note that the pGEM-7Zf(+) plasmid and the herring spermDNA do not include a human viral promoter or enhancer. The promoters inpGEM-7Zf(+) is the bacteriophage SP6 and bacteriophage T7 RNA polymerasepromoters (a bacteriophage is a virus that infects bacteria). Secondnote that a decrease in the size of the insert, increases the copynumber of the insert plasmid resulting in accentuated microcompetitionwith the test plasmid.

[0965] The study also measured the effect of cotransfection on theactivity of the androgen receptor (AR). The study transfected COS-7cells with 20 ng pIRES-AR, pcDNA-AR or pSG5-AR plasmids which expressAR, 500 ng MMTV-luc which highly expresses luc following AR stimulationof the MMTV promoter, and increasing amounts of the empty expressionvector. The pGEM-7Zf(+) plasmid was used instead of the expressionplasmid to maintain a 650 ng final concentration of cotransfected DNA.Transfected cells were treated with 10 nM R1881, an AR ligand, andluciferase activity was measured. The luc activity in the presence of650 ng pGEM-7Zf(+) was arbitrarily set to 1, and the relative lucactivity was calculated. FIG. 8 presents the results (Hofman 2000, ibid,FIG. 5a).

[0966] According to Hofman, et al., (2000, ibid) “The MMTV-luciferaseresponse was strongly reduced in the presence of increasingconcentrations of the empty expression vector and the reduced receptoractivities were proportional to AR expression levels.” The decrease inMMTV-luc transcription resulted from decreased transcription of the ARgene expressed by the pIRES-AR, pcDNA-AR, and pSG5-AR plasmids (see alsoHofman 2000, ibid, FIG. 5b). Transfection with the calcium phosphateprecipitation method, instead of FuGENE-6™, produced similar results.

[0967] Finally, the study transiently cotransfected COS-7 cells with 20ng pSG5-AR, 20 ng pS40-β-galactosidase (βGAL) and increasing amounts ofthe empty pSG5 vector. pGEM-7Zf(+) was used to maintain the DNAconcentration at a constant level. Luc and βGAL activities in thepresence of 650 pGEM-7Zf(+) were arbitrarily set to 1, and the relativeluc activity was calculated. FIG. 9 present the results (Hofman 2000,ibid, FIG. 7a).

[0968] Based on these observations, Hofman, et al., (2000, ibid)concluded that “The most likely explanation is that the total amount oftransfected expression vectors largely exceeds the capacity of thetranscriptional machinery of the cell. For that reason, competitionoccurs between the receptor construct and the cotransfected construct.”

[0969] (10) Choi 2001

[0970] Another study (Choi 2001²³⁰) stably transfected the humanMM-derived cell line ARH with the pcDNA₃ vector carrying an antisense tothe macrophage inflammatory protein 1-α (MIP-1α) (AS-ARH). As control,the study transfected other ARH cells with the “empty” pcDNA₃ vector(EV-ARH). To measure the effect of the antisense on cell growth, thestudy cultured 10⁵ non-transfected (wild type), empty vector, and MIP-1aantisenese (antisense) transfected ARH cells in six-well plates withRPMI-1640 media containing 10% FBS. At days 3 and 5, the cells weresampled, stained and counted. FIG. 10 present the results (Choi 2001,ibid, FIG. 2a).

[0971] After 5 days in culture, the number of cell transfected with theempty vector was larger than the non-transfected cells.

[0972] The study also measured MIP-1α expression in vivo. Wild type,empty vector transfected, and antisense transfected ARH cell wereinfused intravenously into SCID mice (n=10 per group). The mice weresacrificed when they became paraplegic. Femurs and vertebrae wereremoved, and bone marrow plasma was obtained. Expression of hMIP-1α wasmeasured with ELISA kits. The following table summarizes the resultsaccording to data points in Choi 2001 (ibid) FIG. 3a. Wild type Emptyvector P value Femur 193.33 591.20 0.042 Vertebrae 389.44 1031.25 0.059Combined 291.39 786.78 0.012

[0973] Expression of hMIP-1α in mice femur was significantly higherafter infusion with cells transfected with the empty vector relativemice infused with non-transfected cells. In mice vertebrae, theexpression of hMIP-1α was borderline higher in mice infused with thecells transfected with the empty vector relative to mice infused withnon-transfected cells. The combined data from the femur and thevertebrae shows a statistically significant effect of transfection withthe empty vector on MIP-1α expression.

[0974] The pcDNA3 vector carries the cytomegalovirus (CMV) promoter. Theobservations demonstrate the effect of microcompetition with pcDNA3, the“empty” vector, on cell proliferation and on MIP-1α expression in vivo.Note that the pcDNA3 vector carries the cytomegalovirus (CMV) promoter.

[0975] (11) Hu 2001

[0976] Another study (Hu 2001²³¹) measured the efficacy and safety of animmunoconjugate (icon) molecule, composed of a mutated mouse factor VII(mfVII) targeting domain, and the Fc effector domain of an IgG1 Ig(mfVII/Fc icon), with the severe combined immunodeficient (SCID) mousemodel of human prostatic cancer. First, the study injected the SCID mices.c. in both rear flanks with the human prostatic cancer line c4-2. Theinjection resulted in skin tumors. Then, on days 0,3,6,9,12,15,33,36,39,and 42, the study injected into the skin tumor on one flank, either thepcDNA_(3.1)(+) vector carrying the icon (four mice), or the empty vector(four mice). The tumor on the other flank was left uninjected. The studymeasured tumor volume in the injected and non injected flanks. FIG. 11presents the results (Hu 2001, ibid, FIG. 3). ∘ denotes tumors injectedwith the vector encoding the icon, •-uninjected tumors in the icontreated mice, Δ-tumors injected with the empty vector, ▾-uninjectedtumors in the empty vector injected mice.

[0977] The experiment was repeated with the human melanoma line TF2instead of the human prostatic cancer line C4-2. The results arepresented in FIG. 12 (Hu 2001, ibid, FIG. 5)

[0978] In both experiments, injection of the “empty vector” stimulatedtumor growth. Compare tumors injected with empty vector (Δ)anduninjected tumors in the empty vector injected mice (▾).

c) Summary

[0979] The following table summarizes the studies above. Promoter meanspromoter/enhancer. Scholer Mercola Scholer Cherington Adam HigginsGordeladze Awazu Hofman Choi Hu 1984 1985 1986 1988 1996 1996 1997 19982000 2001 2001 Viral Viral SV40 SV40 SV40 CMV MMTV SV40 promoter pro-MSV SV40 CMV effect on moter BK cotransfected RSV (exogenous) geneexpression/ activity Plasmid pSV2CAT pSVCAT pSV2neo pCMV- pZip- pSG5pSV2neo βgal Neo pIRES pSRM2Δ pSV- pcDNA βgal pSV40 Gene SV40 murinehMT-IIA PDGF- HSL SV40 MSV Ig H B CMV BK RSV Viral Viral CMV promoterpro- effect on moter cellular (endog- enous) gene expression/ activityPlasmid pcDNA Gene hMIP- 1α Viral Viral MMTV HSV SV40 CMV CMV promoterpro- MMTV effect on moter cellular function Plasmid pZIP- HSV- pSV40-pcDNA3 pcDNA3 neo neo neo pZIP- neo Func- Cell Cell Cell Cell Tumor tionDiffer- Differ- growth growth growth enti- enti- ation ation Study YesYes Yes No Yes No Yes No Yes No No includes (1) (2) (3) (4) (5) (6) (7)(8) (9) reference to effect of viral promoter (empty vector) Study No NoNo No No No No No No No No includes reference to disease

[0980] References in the study to the effect of the viral promoter(empty vector):

[0981] (1) Scholer 1984: The study measured and discussed competitionbetween different viral enhancers in contransfected studies. Forinstance, the study reports competition between the SV40 and MSVenhancers. The competition between viral enhancers was also observed inHofman 2000 (see above). The study includes no discussion relating theeffect of such competition with endogenous gene expression, cellularfunction, and disease.

[0982] (2) Mercola 1985: The study measured and discussed competitionbetween SV40 enhancer and the cotransfected Ig H enhancer. The studyincludes no discussion relating the effect of such competition withendogenous gene expression, cellular function, and disease.

[0983] (3) Scholer 1986 (ibid): The study measured and discussedcompetition between SV40 enhancer and the cotransfected hMT-IIApromoter. The study includes no discussion relating the effect of suchcompetition with endogenous gene expression, cellular function, anddisease.

[0984] (4) Adam 1996 (ibid): The study measured and discussedmicrocompetition between the CMV and SV40 promoter/enhancer and thecotransfected PDGF-B promoter/enhancer.

[0985] Based on the observations, the study concluded that “Of moregeneral interest, these results indicate that care should be exercisedwhen using commonly available reporter gene constructs to standardizetransfection efficiencies. It is possible that the importance of somepotential gene regulatory sequences could be under estimated, oroverlooked entirely, given certain combinations of reference plasmidco-transfection conditions and cell-types. Moreover, “The results wepresent here indicate a warning note for the use of co-transfectedreference plasmids under the control of viral enhancers: Initialcalibration experiments to determine the appropriate reference plasmidand the optimal relative molar concentration may be worthwhile in orderto avoid erroneous interoperations of such transfection data.” Theauthors interpret the data in the narrow context of laboratorytechniques, specifically, reference plasmids. No relation is suggestedbetween microcompetition, endogenous gene expression, cellular function,or disease.

[0986] (5) Gordeladze 1997 (ibid): The only reference to the differencebetween pre-differentiated and post-differentiated 3T3-L1 cellstransfected with the pZipNeo “empty vector” is the following sentence:“However, post-differentiated vector transfected cells exhibited anon-significant alteration compared to corresponding pre-differentiatedcells.” Note that, although the authors used the term “non-significantalteration,” the paper reports no quantitative analysis of the blot inFIG. 11, and specifically, no statistical analysis that can justify theuse of the term “significant.” Contrary to the authors conclusion, avisual inspection of the blot in FIG. 11 shows a decline of HSL mRNA inthe post-differentiated compared to the pre-differentiated cellstransfected with the empty vector.

[0987] (6) Awazu 1998: The study does not compare between nontransfected(HuH-7 wild) and empty vector transfected (HuH-7 neo) cells. It isinteresting that the study called both the “HuH-7 wild” and “HuH-7 neo”the nontransfected cells. In particular, the study does not mention arelation between microcompetition, endogenous gene expression, cellularfunction, or disease.

[0988] (7) Hofman 2000 (ibid): Measured competition between differentviral enhancers in cotransfected experiments. In the discussion theauthors remark that “Whether this competition occurs at the level oftranscription initiation or at a later step is not clear.” Moreover,based on the observations, the study concluded that “Moreover, it isrecommended to limit the amount of (co)transfected expression plasmidand to avoid the use of empty expression plasmid as a control. Finally,one should be aware of similar misleading results in other experimentalset-ups base on cotransfection.” Similar to Adam 1996 (see above), theauthors interpret the data in the narrow context of laboratory set-ups,specifically, the use of empty vectors as controls in cotransfectionstudies. No relation is suggested between the observed microcompetition,endogenous gene expression, cellular function, or disease.

[0989] (8) Choi 2001: The study includes comparisons between antisensetransfected cells and either empty vector transfected cells or wild typecells as controls. The study does not include a comparison between theempty vector transfected and the wild type cells, that is, between thetwo “controls.”

[0990] (9) Hu 2001: The study does not compare between the tumorsinjected with the control (empty) vector and the uninjected tumors incontrol mice. The only reference to the effect of the empty vector asreported in FIGS. 3 and 5. is the following sentence: “In mice injectedwith the control vector, the tumors on both flanks grew continuously,and the mice died or had to be euthanized by day 57.”

[0991] Conclusion: these studies demonstrate the commitment of theresearch community to the “protein-dependent” paradigm. Each study usedtwo types of plasmids, one with a gene of interest, for instance,cellular Rb or viral T antigen, an another with a reporter gene undercontrol of a viral promoter/enhancer. The second plasmid was considered“empty,” and was, therefore, used as control. All studies above reportobservations which clearly show a significant effect of the “empty”plasmid on gene expression, cell cycle progression, cell proliferationor cell differentiation. However, some of these studies include noreference to these observation, the observations are completely ignored.Moreover, even the studies which discuss the effect of the empty vector,miss the relation between microcompetition and disease.

2. Aberrant Transcription and Disease a) Introduction

[0992] It is a well known fact that aberrant transcription, resultingfrom, for instance, a mutation or hypermethylation, may result indisease. Consider, for instance, the Online Mendelian Inheritance in Man(OMIM™) database which catalogs specific mutations and their associationwith genetic disorders. The following examples demonstrate the effect ofcontrolled mutation in three specific genes, MT, PDGF-B, and HSL on thesubject health.

b) Examples

[0993] (1) MT-I or MT-II Deficiency and Disease (Weight Gain)

[0994] Mice with mutated MT-I and MT-II genes are apparentlyphenotypically normal, despite reduced expression of the metallothioneingenes. The disruption shows no adverse effect on their ability toreproduce and rear offspring. However, after weaning, MT-null miceconsume more food and gain weight at a higher rate compared to controls.The majority of adult male mice in the MT-null colony showed moderateobesity (Beattie 1998²³²). Lead treated MT-null mice showed dose-relatednephromegaly, and following exposure, reduced renal function compared towild type (Qu 2002²³³). MT-I+II knock out (MTKO) mice showed highersusceptibility to autoimmune encephalomyelitis (EAE) compared to wildtype (Penkowa 2001²³⁴), and increased susceptibility to theimmunosuppresseive effects of ultraviolet B radiation and cis-urocanicacid (Reeve 2000²³⁵). MT-I/II null mice also showed increased liver andkidney damage following chronic exposure to inorganic arsenicals (Liu2000¹³⁶).

[0995] (2) PDGF-B Deficiency and Disease

[0996] In mice, a PDGF-B deficiency is embryonic lethal and isassociated with cardiovascular, renal, placental and hematologicaldisorders. Specifically, mice show formation of hemorrhage,microaneurysm, and microvessel leakage. The mice also show lack ofkidney glomerular mesangial cells and microvascular pericytes, andreduced or complete loss of vascular smooth muscle cells (SMC) aroundsmall and medium sized arteries. The mice also show dilated heart andaorta, anemia and thrombocytopenia (Kaminski 2001²³⁷, Lindahl 1997²³⁸).

[0997] (3) HSL Deficiency and Disease (Adipocyte Hypertrophy)

[0998] HSL knockout mice were generated by homologous recombination inembryonic stem cells. Cholesterol ester hydrolase (NCEH) activities werecompletely absent from both brown adipose tissue (BAT) and white adiposetissue (WAT) in mice homozygous for the mutant HSL allele (HSL-/-). Thecytoplasmic area of BAT adipocytes was increased 5-fold in HSL-/-mice(Osuga 2000²³⁹, FIG. 3a) and the median cytoplasmic areas in WAT wasenlarged 2-fold (Ibid, FIG. 3b). The HSL knockout mice showed adipocytehypertrophy. HSL-deficient mice are normoglycemic and normoinsulinemicunder basal conditions. However, after overnight fast, the mice showedreduce concentration of circulating free fatty acids (FFAs) relative tocontrol and heterozygous mice. Moreover, an intraperitoneal glucosetolerance test of the HSL-null mice revealed insulin resistance (Roduit2001²⁴⁰). HSL-deficient male mice are infertile (Chung 2001²⁴¹).HSL-deficient mice also showed other defects associated withmobilization of triglycerides (TG), diglycerides (DG) and cholesterylesters (Haemmerle 2002A²⁴², Haemmerle 2002B²⁴³).

c) Summary

[0999] Microcompetition between a foreign polynucleotide and a cellulargene for a limiting transcription complex result in aberranttranscription of the cellular gene. Aberrant transcription results indisease. Therefore, microcompetition between a foreign polynucleotideand a cellular gene for a limiting transcription complex results indisease. When the foreign polynucleotide persists in the host cell foran extended period of time, microcompetition between the foreignpolynucleotide and the cellular gene results in a chronic disease.

3. Limiting Transcription Factors a) Examples

[1000] The coactivator p300 is a 2,414-amino acid protein initiallyidentified as a binding target of the E1A oncoprotein. cbp is a2,441-amino acid protein initially identified as a transcriptionalactivator bound to phosphorylated cAMP response element (CREB) bindingprotein (hence, cbp). p300 and cbp share 91% sequence identity and arefunctionally equivalent. Both p300 and cbp are members of a family ofproteins collectively referred to as p300/cbp.

[1001] Although p300/cbp are widely expressed, their cellularavailability is limited. Several studies demonstrated inhibitedactivation of certain transcription factors resulting from competitivebinding of p300/cbp to other cellular or viral proteins. For example,competitive binding of p300 or CBP to the glucocorticoid receptor (GR),or retinoic acid receptor (RAR), inhibited activation of a promoterdependent on the AP-1 transcription factor (Kamei 1996²⁴⁴). Competitivebinding of cbp to STAT1α inhibited activation of a promoter dependent onboth the AP-1 and ets transcription factors (Horvai 1997²⁴⁵).Competitive binding of p300 to STAT2 inhibited activation of a promoterdependent on the NF-κB RelA transcription factor (Hottiger 1998²⁴⁶).Other studies also demonstrated limited availability of p300/cbp, see,for instance, Pise-Masison 2001²⁴⁷, Banas 2001²⁴⁸, Wang 2001²⁴⁹, Ernst2001²⁵⁰, Yuan 2001²⁵¹, Ghosh 2001²⁵², Li 2000²⁵³, Nagarajan 2000²⁵⁴,Speir 2000²⁵⁵, Chen 2000²⁵⁶, and Werner 2000²⁵⁷.

4. Transcription Factors Microcompeted by Foreign Polynucleotides a)Examples

[1002] One example of a foreign polynucleotide typically found in hostcells is viral DNA. Several cellular transcription factors formcomplexes on viral DNA, and transactivate or suppress viraltranscription. Consider GA binding protein (GABP), also called NuclearRespiratory Factor 2 (NRF-2)²⁵⁸, E4 Transcription factor 1 (E4TF1)²⁵⁹,and Enhancer Factor 1 A (EF-1A)²⁶⁰, as an example. The literature listsfive subunits of GABP: GABPα, GABPβ1, GABPβ2 (together called GABPβ),GABPγ1 and GABPγ2 (together called GABPγ). GABPα is an ets-relatedDNA-binding protein which binds the DNA motif (A/C)GGA(A/T)(G/A), termedthe N-box. GABPα forms a heterocomplex with GABPβ which stimulatestranscription efficiently both in vitro and in vivo. GABPα also forms aheterocomplex with GABPγ, but the heterodimer does not stimulatetranscription. The degree of transactivation by GABP appears tocorrelate with the relative intracellular concentrations of GABPβ andGABPγ. An increase in GABPβ relative to GABPγ increases transcription,while an increase of GABPγ relative to GABPβ decreases transcription.The degree of transactivation by GABP is, therefore, a function of theratio between GABPβ and GABPγ. Control of this ratio within the cellregulates transcription of genes with binding sites for GABP (Suzuki1998²⁶¹).

[1003] The N-box is the core binding sequence of many viral enhancersincluding the polyomavirus enhancer area 3 (PEA3) (Asano 1990²⁶²),adenovirus E1A enhancer (Higashino 199²⁶³), Rous Sarcoma Virus (RSV)enhancer (Laimins 1984²⁶⁴), Herpes Simplex Virus 1 (HSV-1) (in thepromoter of the immediate early gene ICP4) (LaMarco 1989²⁶⁵, Douville1995²⁶⁶), Cytomegalovirus (CMV) (IE-1 enhancer/promoter region) (Boshart1985²⁶⁷), Moloney Murine Leukemia Virus (Mo-MuLV) enhancer (Gunther1994²⁶⁸), Human Immunodeficiency Virus (HIV) (the two NF-κB bindingmotifs in the HIV LTR) (Flory 1996²⁶⁹), Epstein-Barr virus (EBV) (20copies of the N-box in the +7421/+8042 oriP/enhancer) (Rawlins 1985²⁷⁰)and Human T-cell lymphotropic virus (HTLV) (8 N-boxes in the enhancer(Mauclere 1995²⁷¹) and one N-box in the LTR (Komfeld 1987²⁷²)). Notethat some viral enhancers, for example SV40, lack a precise N-box, butstill bind the GABP transcription factor (Bannert 1999²⁷³).

[1004] Ample evidence exists supporting binding of GABP to the N-boxesin these viral enhancers. For instance, Flory, et al., (1996²⁷⁴) showedbinding of GABP to the HIV LTR, Douville, et al., (1995²⁷⁵) showedbinding of GABP to the promoter of ICP4 of HSV-1, Bruder, et al.,(1991²⁷⁶) and Bruder, et al., (1989²⁷⁷) showed binding of GABP to theadenovirus E1A enhancer element I, Ostapchuk, et al., (1986²⁷⁸) showedbinding of GABP (called EF-1A in their paper) to the polyomavirusenhancer and Gunther, et al., (1994²⁷⁹) showed binding of GABP toMo-MuLV. Other studies demonstrate competition between the above viralenhancers and enhancers of other viruses. Scholer and Gruss, (1984²⁸⁰)showed competition between the Moloney Sarcoma Virus (MSV) enhancer andSV40 enhancer and competition between the RSV enhancer and the BK virusenhancer.

[1005] Other cellular transcription factors also form complexes on viralDNA, and transactivate or suppress viral transcription. For instance,AML1 binds the polyomavirus (Chen 1998²⁸¹), Mo-MLV (Lewis 1999²⁸², Sun1995²⁸³), and SL3 retrovirus (Martiney 1999A²⁸⁴, Martiney 1999B²⁸⁵),NF-AT binds HIV-1 (NFAT1 binds the NF-κKB site in the viral LTR) (Cron2000²⁸⁶), HNF4α binds the Hepatitis B virus (Wang 1998²⁸⁷), theSmad3/Smad4 complex binds the Epstein-Barr virus (Liang 2000²⁸⁸), ets1binds the human cytomegalovirus (Chen 2000²⁸⁹), NF-YB binds the humancytomegalovirus (Huang 1994²⁹⁰), hepatitis B virus (Lu 1996²⁹¹, Bock1999²⁹²), minute virus (Gu 1995²⁹³), adenovirus (Song 1998²⁹⁴), andvaricella-zoster virus (Moriuchi 1995²⁹⁵), ATF-2 binds the human T-cellleukemia type 1 (HTLV-I) (Xu 1996²⁹⁶, Xu 1994²⁹⁷), and hepatitis B virus(Choi 1997²⁹⁸), p53 binds the polyomavirus (Py) (Kanda 1994²⁹⁹), humanCMV (Allamane 2001³⁰⁰, Deb 2001³⁰¹), human immunodeficiency virus type 1(HIV-1) (Deb 2001, ibid), and the Hepatitis B virus (Lee 1998³⁰², Ori1998³⁰³), YY-1 binds the human papillomavirus type 18 (HPV-18) (Jundt1995³⁰⁴), NF-kB binds HIV (Hottiger 1998, ibid), Stat2 binds HIV(Hottiger 1998, ibid), and C/EBβ binds the Hepatitis B virus (Lai1999³⁰⁵, Gilbert 2000³⁰⁶), and HIV-1 (LTR) (Honda 1998³⁰⁷), and theglucocorticoid receptor (GR) binds the mouse mammary tumor virus LTR(Pfitzner 1998, ibid).

[1006] Note that all the above mentioned transcription factors bind thelimiting coactivator p300/cbp (Bannert 1999³⁰⁸, Kitabayashi 1998³⁰⁹,Garcia-Rodriguez 1998³¹⁰, Sisk 2000³¹¹, Soutoglou 2000³¹², Janknecht1998³¹³, Feng 1998³¹⁴, Pouponnot 1998³¹⁵, Jayaraman 1999³¹⁶, Li 1998³¹⁷,Duyndam 1999³¹⁸, Avantaggiati 1997³¹⁹ Van Order 1999³²⁰, Hottiger1998³²¹, Gerritsen 1997³²², Hottiger 1998, ibid, Paulson 1999, ibid,Gringras 1999, ibid, Bhattacharya 1996³²³, Mink 1997³²⁴, Pfitzner 1998,ibid). Since p300/cbp is limiting, a transcription complex that includesp300/cbp is also limiting. For instance, since p300/cbp is limiting,GABPop•300/cbp is also limiting.

We claim:
 1. A method for the treatment of a chronic disease, comprisingadministrating to an animal or human subject an effective amount of anagent which modifies microcompetition between a polynucleotide naturalto said subject and a polynucleotide foreign to said subject.
 2. Themethod of claim 1, wherein said polynucleotide foreign to said subjectis a viral promoter.
 3. The method of claim 1, wherein saidpolynucleotide foreign to said subject is a viral enhancer.
 5. A methodfor the treatment of a chronic disease, comprising administrating to ananimal or human subject an effective amount of an agent which modifiesthe complex between a transcription factor natural to said subject and apolynucleotide foreign to said subject.
 6. The method of claim 5,wherein said polynucleotide foreign to said subject is a viral promoter.7. The method of claim 5, wherein said polynucleotide foreign to saidsubject is a viral enhancer.
 9. A method for the treatment of a chronicdisease, comprising administrating to an animal or human subject aneffective amount of an agent which modifies the complex between atranscription factor and a polynucleotide, where said transcriptionfactor and polynucleotide are natural to the same said subject, andwhere said polynucleotide is susceptible to microcompetition with apolynucleotide foreign to said subject.
 10. The method of claim 9,wherein said polynucleotide foreign to said subject is a viral promoter.11. The method of claim 9, wherein said polynucleotide foreign to saidsubject is a viral enhancer.
 13. A method for the treatment of a chronicdisease, comprising administrating to an animal or human subject aneffective amount of an agent which modifies the expression of a gene, orgene fragment, where said gene is natural to said subject, and said geneis susceptible to microcompetition with a polynucleotide foreign to saidsubject.
 14. The method of claim 13, wherein said polynucleotide foreignto said subject is a viral promoter.
 15. The method of claim 13, whereinsaid polynucleotide foreign to said subject is a viral enhancer.
 17. Amethod for the treatment of a chronic disease, comprising administratingto an animal or human subject an effective amount of an agent whichmodifies the activity of a gene product of a gene, or gene fragment,where said gene is natural to said subject, and said gene is susceptibleto microcompetition with a polynucleotide foreign to said subject. 18.The method of claim 17, wherein said polynucleotide foreign to saidsubject is a viral promoter.
 19. The method of claim 17, wherein saidpolynucleotide foreign to said subject is a viral enhancer.
 21. A methodfor the treatment of a chronic disease, comprising administrating to ananimal or human subject an effective amount of an agent which modifiesthe copy number of a polynucleotide foreign to said subject, in a saidsubject cell.
 22. The method of claim 21, wherein said polynucleotideforeign to said subject is a viral promoter.
 23. The method of claim 21,wherein said polynucleotide foreign to said subject is a viral enhancer.25. A method for the treatment of a chronic disease, comprisingadministrating to an animal or human subject an effective amount of anagent which modifies the copy number of a latent polynucleotide foreignto said subject, in a said subject cell.
 26. The method of claim 25,wherein said polynucleotide foreign to said subject is a viral promoter.27. The method of claim 25, wherein said polynucleotide foreign to saidsubject is a viral enhancer.